Guanidine-containing compounds useful as muscarinic receptor antagonists

ABSTRACT

The invention provides compounds of formula I: 
                         
or a pharmaceutically acceptable salt thereof, wherein R 1-3 , R 5-7 , a, X, Y, Y′, Y″, and Z are as defined in the specification. These compounds are muscarinic receptor antagonists. The invention also provides pharmaceutical compositions containing such compounds, processes for preparing such compounds and methods of using such compounds to, for example, treat pulmonary disorders such as chronic obstructive pulmonary disease and asthma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.13/468,200, filed on May 10, 2012, now allowed, which is a continuationapplication of U.S. Ser. No. 13/228,782, filed on Sep. 9, 2011, andissued as U.S. Pat. No. 8,198,304, which is a divisional application ofU.S. Ser. No. 13/096,651, filed on Apr. 28, 2011 and issued as U.S. Pat.No. 8,039,489, which is a divisional application of U.S. Ser. No.12/231,861, filed on Sep. 5, 2008, and issued as U.S. Pat. No.7,960,385, which claims the benefit of U.S. Provisional Application No.60/967,914, filed on Sep. 7, 2007; the disclosures of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to guanidine-containing compounds havingmuscarinic receptor antagonist or anticholinergic activity. Theinvention also relates to pharmaceutical compositions comprising thesecompounds, processes for preparing them and methods of use to treatpulmonary disorders.

2. State of the Art

Pulmonary or respiratory disorders, such as chronic obstructivepulmonary disease (COPD) and asthma, afflict many millions of peopleworldwide and such disorders are a leading cause of morbidity andmortality.

Muscarinic receptor antagonists are known to provide bronchoprotectiveeffects and therefore, such compounds are useful for treatingrespiratory disorders, such as COPD and asthma. When used to treat suchdisorders, muscarinic receptor antagonists are typically administered byinhalation. However, even when administered by inhalation, a significantamount of the muscarinic receptor antagonist is often absorbed into thesystemic circulation resulting in systemic side effects, such as drymouth, mydriasis and cardiovascular side effects.

Additionally, many inhaled muscarinic receptor antagonists have arelatively short duration of action requiring that they be administeredseveral times per day. Such a multiple-daily dosing regime is not onlyinconvenient but also creates a significant risk of inadequate treatmentdue to patient non-compliance with the required frequent dosingschedule.

Accordingly, a need exists for new muscarinic receptor antagonists. Inparticular, a need exists for muscarinic receptor antagonists havinghigh potency, reduced systemic side effects when administered byinhalation, and a long duration of action thereby allowing foronce-daily or even once-weekly dosing. In addition, a need exists formuscarinic receptor antagonists having high affinity for the receptorand a long receptor half life. Such compounds are expected to beparticularly effective for treating pulmonary disorders, such as COPDand asthma, while reducing or eliminating side effects, such asdry-mouth and constipation.

SUMMARY OF THE INVENTION

The present invention provides novel guanidine-containing compoundswhich have muscarinic receptor antagonist or anticholinergic activity.Among other properties, compounds of this invention have been found topossess improved binding affinity for hM₂ and hM₃ muscarinic receptorsubtypes, have longer receptor half-lives, have a larger therapeuticwindow, or have greater potency compared to related compounds.Accordingly, compounds of the invention are expected to be useful andadvantageous as therapeutic agents for treating pulmonary disorders.

One aspect of the invention relates to compounds having formula I:

wherein:

R¹ is selected from —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₃₋₉cycloalkyl, andheteroaryl; R² is selected from aryl and heteroaryl; R³ is selected fromH and —C₀₋₁alkylene-OH, or R³ forms a double bond with R¹; or —CR¹R²R³together form a group of formula:

where A is selected from a bond, —O—, —S—, —CH₂—, —CH═CH—, —CH₂CH₂—,—NH—, and —N(CH₃)—; and R⁴ is selected from H, halo, —OH, —C₁₋₈alkyl,and —C₁₋₈alkoxy;

X is selected from a bond, —O—, and —O—CH₂—; when X is a bond, Y is—CH₂—, Y′ is —N—, and Y″ is —CH₂—; and when X is —O— or —O—CH₂—, Y′ is—CH—, Y is a bond and Y″ is —CH₂— or —(CH₂)₂—, or Y is —CH₂— and Y″ is—CH₂—;

R⁵ is selected from fluoro and —C₁₋₄alkyl; and a is 0 or an integer offrom 1 to 3;

R⁶ and R⁷ are independently selected from H and —C₁₋₄alkyl, and furtherwherein one of R⁶ or R⁷ may be —NH₂;

Z is selected from H, —C₁₋₆allyl, —C₁₋₃alkylene-Q, and—NH—C₀₋₁alkylene-Q; Q is selected from —C₃₋₇cycloalkyl, aryl, andheteroaryl; and Q is optionally substituted with 1-5 R⁸ groupsindependently selected from halo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano,—C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl, —CONR^(8a)R^(8b),—NH—C(O)—C₁₋₄alkyl, —N-di-C₁₋₄alkyl, and —N⁺(O)O; R^(8a) and R^(8b) areindependently selected from H and —C₁₋₄alkyl;

wherein R¹ and R² are optionally substituted with 1 to 5 R^(a) groupsindependently selected from —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl,—C₃₋₆cycloalkyl, cyano, halo, —OR^(b), —C(O)OR^(b), —SR^(b), —S(O)R^(b),—S(O)₂R^(b), —C(O)NR^(c)R^(d), and —NR^(c)R^(d); each R^(b) isindependently selected from H, —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl,and —C₃₋₆cycloalkyl; each R^(c) and R^(d) is independently selected fromH, —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, and —C₃₋₆cycloalkyl;

wherein each alkyl, alkenyl, alkynyl, alkylene, and cycloalkyl group inR^(a-d), R⁴⁻⁸, and Z, is optionally substituted with 1 to 5 fluoroatoms; wherein each cycloalkyl in R^(a-d) is optionally substituted with1 to 3 substituents independently selected from —C₁₋₄alkyl,—C₂₋₄alkenyl, —C₂₋₄alkynyl, cyano, halo, —O(C₁₋₄alkyl), —S(C₁₋₄alkyl),—S(O)(C₁₋₄alkyl), —S(O)₂(C₁₋₄alkyl), —NH₂, —NH(C₁₋₄alkyl), and—N(C₁₋₄alkyl)₂, wherein each alkyl, alkenyl and alkynyl group isoptionally substituted with 1 to 5 fluoro substituents; and the alkylenegroup in Z is optionally substituted with 1 or 2 substituentsindependently selected from —C₁₋₂alkyl and —OH; or a pharmaceuticallyacceptable salt thereof.

Among the compounds of formula I, compounds of particular interest arethose having an inhibition dissociation constant (K_(i)) for binding tothe M₃ receptor subtype of less than or equal to 100 nM; in particularhaving a K_(i) less than or equal to 50 nM; more particularly having aK_(i) less than or equal to 10 nM; and even more particularly having aK_(i) less than or equal to 1.0 nM.

Another aspect of the invention relates to pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a compound of theinvention. Such compositions may optionally contain other therapeuticagents such as steroidal anti-inflammatory agents (e.g.,corticosteroids), β₂ adrenergic receptor agonists, phosphodiesterase-4inhibitors, and combinations thereof. Accordingly, in yet another aspectof the invention, a pharmaceutical composition comprises a compound ofthe invention, a second active agent, and a pharmaceutically acceptablecarrier. Another aspect of the invention relates to a combination ofactive agents, comprising a compound of the invention and a secondactive agent. The compound of the invention can be formulated togetheror separately from the additional agent(s). When formulated separately,a pharmaceutically acceptable carrier may be included with theadditional agent(s). Thus, yet another aspect of the invention relatesto a combination of pharmaceutical compositions, the combinationcomprising: a first pharmaceutical composition comprising a compound ofthe invention and a first pharmaceutically acceptable carrier; and asecond pharmaceutical composition comprising a second active agent and asecond pharmaceutically acceptable carrier. This invention also relatesto a kit containing such pharmaceutical compositions, for example wherethe first and second pharmaceutical compositions are separatepharmaceutical compositions.

Compounds of the invention possess muscarinic receptor antagonistactivity, and are therefore expected to be useful as therapeutic agentsfor treating patients suffering from a disease or disorder that istreated by blocking the muscarinic receptor. Thus, one aspect of theinvention relates to a method of producing bronchodilation in a patient,comprising administering to the patient a bronchodilation-producingamount of a compound of the invention. The invention is also directed tomethod of treating a pulmonary disorder such as chronic obstructivepulmonary disease or asthma, comprising administering to a patient atherapeutically effective amount of a compound of the invention. Anotheraspect of the invention relates to a method for antagonizing amuscarinic receptor in a mammal comprising administering to the mammal,a muscarinic receptor-antagonizing amount of a compound of theinvention.

Since compounds of the invention possess muscarinic receptor antagonistactivity, such compounds are also useful as research tools. Accordingly,one aspect of the invention relates to a method of using a compound ofthe invention as a research tool, the method comprising conducting abiological assay using a compound of the invention. Compounds of theinvention can also be used to evaluate new chemical compounds. Thusanother aspect of the invention relates to a method of evaluating a testcompound in a biological assay, comprising: (a) conducting a biologicalassay with a test compound to provide a first assay value; (b)conducting the biological assay with a compound of the invention toprovide a second assay value; wherein step (a) is conducted eitherbefore, after or concurrently with step (b); and (c) comparing the firstassay value from step (a) with the second assay value from step (b).Exemplary biological assays include a muscarinic receptor binding assayand a bronchoprotection assay in a mammal. Still another aspect of theinvention relates to a method of studying a biological system or samplecomprising a muscarinic receptor, the method comprising: (a) contactingthe biological system or sample with a compound of the invention; and(b) determining the effects caused by the compound on the biologicalsystem or sample.

The invention is also directed to processes and intermediates useful forpreparing compounds of the invention. Accordingly, another aspect of theinvention relates to a process of preparing compounds of the invention,comprising: (a) coupling compound (1) and compound (2) under amidebond-forming conditions and deprotecting the product to form compound(3), or Mitsunobu coupling or transesterification of compound (1) andcompound (4) and deprotecting the product to form compound (5); (b)reacting compound (3) or compound (5) with compound (6) to form compound(7); and (c) reacting compound (7) and compound (8) to provide acompound of formula I; wherein compounds (1) through (9) are as definedherein. In other aspects, the invention relates to products prepared byany of the processes described herein.

Yet another aspect of the invention relates to the use of a compound ofthe invention for the manufacture of a medicament, especially for themanufacture of a medicament useful for treating a pulmonary disorder orfor antagonizing a muscarinic receptor in a mammal. Still another aspectof the invention relates to the use of a compound of the invention as aresearch tool. Other aspects and embodiments of the invention aredisclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, this invention relates to compounds having formula I:

or a pharmaceutically acceptable salt thereof. This formula may also bedepicted as:

As used herein, the term “compound of the invention” includes allcompounds encompassed by formula I such as the species embodied informulas II-VIII. In addition, when the compound of the inventioncontain a basic or acidic group (e.g., amino or carboxyl groups), thecompound can exist as a free base, free acid, or in various salt forms.All such salt forms are included within the scope of the invention.Accordingly, those skilled in the art will recognize that reference to acompound herein, for example, reference to a “compound of the invention”or a “compound of formula I” includes a compound of formula I as well aspharmaceutically acceptable salts of that compound unless otherwiseindicated. Furthermore, solvates of compounds of formula I are includedwithin the scope of the invention.

The compounds of the invention may contain one or more chiral centersand so may exist in a number of stereoisomeric forms. When such chiralcenters are present, this invention relates to racemic mixtures, purestereoisomers (i.e., enantiomers or diastereomers),stereoisomer-enriched mixtures, and the like unless otherwise indicated.When a chemical structure is depicted without any stereochemistry, it isunderstood that all possible stereoisomers are encompassed by suchstructure. Thus, for example, the term “compound of formula I” isintended to include all possible stereoisomers of the compound.Similarly, when a particular stereoisomer is shown or named herein, itwill be understood by those skilled in the art that minor amounts ofother stereoisomers may be present in the compositions of this inventionunless otherwise indicated, provided that the utility of the compositionas a whole is not eliminated by the presence of such other isomers.Individual enantiomers may be obtained by numerous methods that are wellknown in the art, including chiral chromatography using a suitablechiral stationary phase or support, or by chemically converting theminto diastereomers, separating the diastereomers by conventional meanssuch as chromatography or recrystallization, then regenerating theoriginal enantiomers. Additionally, where applicable, all cis-trans orE/Z isomers (geometric isomers), tautomeric forms and topoisomeric formsof the compounds of this invention are included within the scope of thisinvention unless otherwise specified.

In particular, the compounds of formula I contain a chiral center at thecarbon atom indicated by the symbol * in the following partial formula(shown without optional substituents for clarity), illustrated with theR¹ C₅₋₉cycloalkyl moiety being cyclopentyl, the R² aryl moiety beingphenyl, and R³ being —OH:

In one embodiment of this invention, the carbon atom identified by thesymbol * has the (R) configuration. In this embodiment, compounds offormula I have the (R) configuration at the carbon atom identified bythe symbol * or are enriched in a stereoisomeric form having the (R)configuration at this carbon atom. In another embodiment, the carbonatom identified by the symbol * has the (S) configuration. In thisembodiment, compounds of formula I have the (S) configuration at thecarbon atom identified by the symbol * or are enriched in astereoisomeric form having the (S) configuration at this carbon atom.

The compounds of the invention, as well as those compounds used in theirsynthesis, may also include isotopically-labeled compounds, i.e., whereone or more atoms have been enriched with atoms having an atomic massdifferent from the atomic mass predominately found in nature. Examplesof isotopes that may be incorporated into the compounds of formula I,for example, include, but are not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸Oand ¹⁷O.

The compounds of the invention have been found to possess muscarinicreceptor antagonist activity. Among other properties, compounds of theinvention have been found to possess improved binding affinity for hM₂and hM₃ muscarinic receptor subtypes, have longer receptor half-lives,and have greater potency compared to related compounds, and are expectedto be useful as therapeutic agents for treating pulmonary disorders.

The nomenclature used herein to name the compounds of the invention isillustrated in the Examples herein. This nomenclature has been derivedusing the commercially-available AutoNom software (MDL, San Leandro,Calif.).

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments of theinvention. These representative values are intended to further defineand illustrate such aspects and embodiments and are not intended toexclude other embodiments or to limit the scope of the invention. Inthis regard, the representation that a particular value or substituentis preferred is not intended in any way to exclude other values orsubstituents from the invention unless specifically indicated.

R¹ may be a —C₁₋₆alkyl, —C₂₋₆alkenyl, —C₃₋₉cycloalkyl or heteroarylgroup that is unsubstituted or substituted with 1 to 5 R^(a) groups.R^(a) is independently selected from —C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl, —C₃₋₆cycloalkyl, cyano, halo, —OR^(b), —C(O)OR^(b),—SR^(b), —S(O)R^(b), —S(O)₂R^(b), —C(O)NR^(c)R^(d), and —NR^(c)R^(d).Each R^(b) is independently selected from H, C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl, and —C₃₋₆cycloalkyl. Each R^(c) and R^(d) group isindependently selected from H, —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl,and —C₃₋₆cycloalkyl. In one embodiment, R¹ is —C₃₋₉cycloalkyl; inanother embodiment —C₃₋₆cycloalkyl, i.e., cyclopropyl, cyclobutyl,cyclopentyl or cyclohexyl; and in yet another embodiment R¹ is—C₅cycloalkyl, i.e., cyclopentyl. In one embodiment, R¹ is a —C₁₋₆alkylgroup such as —CH₂CH(CH₃)₂. In another embodiment, R¹ is a —C₂₋₆alkenylgroup such as —CH₂CHCH₂. In one embodiment, R¹ is unsubstituted. Inanother embodiment, R¹ is a heteroaryl, such as thiophenyl (includingthiophen-2-yl and thiophen-3-yl).

Each alkyl, alkenyl, alkynyl, alkylene, and cycloalkyl group in R^(a),R^(b), R^(c), and R^(d), may be substituted with 1 to 5 fluoro atoms. Inaddition, each cycloalkyl in R^(a-d) may be substituted with 1 to 3substituents independently selected from —C₁₋₄alkyl, —C₂₋₄alkenyl,—C₂₋₄alkynyl, cyano, halo, —O(C₁₋₄alkyl), —S(C₁₋₄alkyl), —S(O)(C₁₋₄alkyl), —S(O)₂ (C₁₋₄alkyl), —NH₂, —NH(C₁₋₄alkyl) and —N(C₁₋₄alkyl)₂,wherein each alkyl, alkenyl and alkynyl group is optionally substitutedwith 1 to 5 fluoro substituents.

R² may be an aryl group that is unsubstituted or substituted with 1 to 5R^(a) groups, which are defined above. In one embodiment, R² is phenyl.In another embodiment, R² is unsubstituted phenyl. In anotherembodiment, R² is a heteroaryl, such as thiophenyl (includingthiophen-2-yl and thiophen-3-yl).

R³ may be H or —C₀₋₁alkylene-OH, or may form a double bond with R¹,which can be depicted as:

In one particular embodiment, R³ is —OH. In addition, —CR¹R²R³ togethermay form a group of formula:

where A is a bond, —O—, —S—, —CH₂—, —CH═CH—, —CH₂CH₂—, —NH—, or—N(CH₃)—, and R⁴ is selected from H, halo, —OH, —C₁₋₈alkyl, and—C₁₋₈alkoxy. The alkyl group in R⁴ may be substituted with 1 to 5 fluoroatoms. In one particular embodiment, —CR¹R²R³ together form:

In this embodiment, A is —O— and R⁴ is H, as shown.

In one embodiment, X is a bond, Y is —CH₂—, Y′ is —N—, and Y″ is —CH₂—,which can be depicted as:

In another embodiment, when X is —O— or —O—CH₂—, Y′ is —CH—, Y is a bondand Y″ is —CH₂— or —(CH₂)₂—, which can be depicted as:

respectively. In another embodiment, when X is —O— or —O—CH₂—, Y′ is—CH—, and both Y and Y″ are —CH₂—, which can be depicted as:

R⁵ is selected from fluoro and —C₁₋₄alkyl. The value for a is 0 or aninteger of from 1 to 3. In one particular embodiment, a is 0. The alkylgroup in R⁵ may be substituted with 1 to 5 fluoro atoms.

R⁶ and R⁷ are independently selected from H and —C₁₋₄alkyl. In addition,one of R⁶ or R⁷ may be —NH₂. In one particular embodiment, R⁶ ishydrogen or —C₁₋₄alkyl. In another embodiment, R⁷ is hydrogen. In yetanother particular embodiment, both R⁶ and R⁷ are hydrogen. The alkylgroup in R⁶ and R⁷ may be substituted with fluoro atoms. For example, R⁶and/or R⁷ can be —CH₃ as well as —CFH₂, —CF₂H or —CF₃.

Z is selected from hydrogen, —C₁₋₆alkyl, —C₁₋₃alkylene-Q, and—NH—C₀₋₁alkylene-Q. In one embodiment, Z is —CH₂-Q. In anotherembodiment, Z is —(CH₂)₂-Q. In still another embodiment, Z is —(CH₂)₃-Q.In still another embodiment, Z is —NH-Q. In yet another embodiment, Z is—NH—CH₂-Q. In another embodiment, Z is hydrogen or —C₁₋₆alkyl. Exemplary—C₁₋₆alkyl groups include methyl, propyl, butyl, and pentyl. The alkyland alkylene groups in Z may be substituted with 1 to 5 fluoro atoms.Further, the alkylene group in Z may be substituted with 1 or 2substituents independently selected from —C₁₋₂alkyl and —OH. Forexample, in one embodiment, Z is —CH(CH₃)—.

Q is a —C₃₋₇cycloalkyl, aryl, or heteroaryl group. Exemplary—C₃₋₇cycloalkyl groups include cyclopropyl, cyclohexyl, and cycloheptyl.Exemplary aryl groups include phenyl and naphthyl. In one embodiment, Qis phenyl. Exemplary heteroaryl groups include pyrrolyl, imidazolyl,thiazolyl, oxazolyl, furanyl, thiophenyl, triazolyl, pyrazolyl,isoxazolyl, isothiazolyl, pyridinyl, pyrazinyl, pyridazinyl,pyrimidinyl, triazinyl, indolyl, benzofuranyl, benzopyranyl,benzothiophenyl, benzoimidazolyl, benzothiazolyl, benzodioxolyl,quinolyl, isoquinolyl, quinazolinyl, and quinoxalinyl groups. Ofparticular interest are thiazolyl (e.g., thiazol-2-yl and thiazol-4-yl),furanyl (e.g., furan-2-yl and furan-3-yl), thiophenyl (e.g.,thiophen-2-yl and thiophen-3-yl), pyrazolyl (e.g., 1H-pyrazol-3-yl),pyridinyl (e.g., pyridin-2-yl), indolyl (e.g., 1H-indol-2-yl,1H-indol-4-yl and 1H-indol-5-yl), benzofuranyl (e.g., benzofuran-5-yl),benzothiophenyl (e.g., benzo[b]thiophen-2-yl and benzo[b]thiophen-5-yl),and benzodioxolyl (e.g., benzo[1,3]dioxol-5-yl) groups.

Q may be substituted with 1 to 5 R⁸ groups independently selected fromhalo (e.g., Cl and F), —C₁₋₄alkyl (e.g., —CH₃), —C₀₋₄alkylene-OH (e.g.,—OH and —CH₂OH), cyano, —C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl (e.g.,—C(O)O—CH₃), —O—C₁₋₄alkyl (e.g., —OCH₃), —S—C₁₋₄alkyl (e.g., —S—CH₃),—CONR^(8a)R^(8b), —NH—(O)—C₁₋₄alkyl, —N-di-C₁₋₄alkyl, and —N⁺(O)O, whereR^(8a) and R^(8b) are independently selected from H and —C₁₋₄alkyl. Eachalkyl and alkylene group in R⁸ may be substituted with 1 to 5 fluoroatoms. For example, R⁸ can be a fluoro substituted —C₁₋₄alkyl group suchas —CF₃ or a fluoro substituted —O—C₁₋₄alkyl group such as —OCF₃.

In one embodiment, Q is substituted with one R⁸ group selected fromhalo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano, —C(O)O—C₁₋₄alkyl,—O—C₁₋₄alkyl, —S—C₁₋₄alkyl, and —CONR^(8a)R^(8b), where each alkyl groupis optionally substituted with 1 to 3 fluoro atoms. In anotherembodiment, Q is substituted with two R⁸ groups that are halo groups(which may be the same or different). In one embodiment, Q is anunsubstituted —C₃₋₇cycloalkyl group. In one embodiment, Q is anunsubstituted aryl group. In another embodiment, Q is an aryl grouphaving one R⁸ group selected from halo, —C₁₋₄alkyl, cyano,—C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl,—CONR^(8a)R^(8b), where each alkyl group is optionally substituted with1 to 3 fluoro atoms. In yet another embodiment, Q is an aryl grouphaving two R⁸ groups that are halo groups. In one embodiment, Q is anunsubstituted heteroaryl group. In another embodiment, Q is a heteroarylgroup having one R⁸ group that is a —C₁₋₄alkyl group.

In one embodiment, the invention relates to compounds having formula I,where R¹ is isobutyl, cyclopentyl, or thiophenyl; R² is phenyl orthiophenyl; R³ is —OH; or —CR¹R²R³ together form a group of formula:

a is 0; R⁶ is H or —C₁₋₄alkyl; R⁷ is H; Z is H, —C₁₋₆alkyl,—C₁₋₃alkylene-Q, or —NH—C₀₋₁alkylene-Q; Q is cyclohexyl, cycloheptyl,phenyl, benzodioxolyl, benzofuranyl, benzothiophenyl, furanyl, indolyl,pyrazolyl, pyridinyl, thiazolyl, or thiophenyl; Q is optionallysubstituted with 1-2 R⁸ groups independently selected from halo,—C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano, —C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl,—S—C₁₋₄alkyl, and —CONH₂; and the alkyl groups in R⁸ are optionallysubstituted with 1 to 5 fluoro atoms.

In another embodiment, the invention relates to a compound havingformula II:

or a pharmaceutically acceptable salt thereof, where R¹⁻³, R⁶⁻⁷, and Zare as defined for formula I. In one particular embodiment, theinvention relates to compounds of formula II, where: R¹ is cyclopentylor thiophenyl; R² is phenyl or thiophenyl; R³ is —OH; R⁶ is H or—C₁₋₂alkyl; R⁷ is H; Z is —C₁₋₆alkyl, —C₁₋₃alkylene-Q, or—NH—C₀₋₁alkylene-Q; Q is cyclohexyl, cycloheptyl, phenyl, benzodioxolyl,benzofuranyl, benzothiophenyl, furanyl, indolyl, pyrazolyl, pyridinyl,thiazolyl, or thiophenyl; Q is optionally substituted with 1-2 R⁸ groupsindependently selected from halo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano,—C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl, —S—C₁₋₄alkyl, and —CONH₂; and the alkylgroups in R⁸ are optionally substituted with 1 to 5 fluoro atoms.

Yet another aspect of the invention relates to compounds having formulaIIa:

or a pharmaceutically acceptable salt thereof, where R⁶ and Z are asdefined for formula I. In one particular embodiment, the inventionrelates to compounds of formula IIa, where: R⁶ is H or —C₁₋₂alkyl; Z is—C₁₋₆alkyl, —C₁₋₃alkylene-Q, or —NH—C₀₋₁alkylene-Q; Q is cyclohexyl,cycloheptyl, phenyl, benzodioxolyl, benzofuranyl, benzothiophenyl,furanyl, indolyl, pyrazolyl, pyridinyl, thiazolyl, or thiophenyl; Q isoptionally substituted with 1-2 R⁸. groups independently selected fromhalo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano, —C(O)O—C₁₋₄alkyl,—O—C₁₋₄alkyl, —S—C₁₋₄alkyl, and —CONH₂; and the alkyl groups in R⁸ areoptionally substituted with 1 to 5 fluoro atoms.

Still another aspect of the invention relates to compounds havingformula IIb:

or a pharmaceutically acceptable salt thereof, where Q is as defined forformula I. In one particular embodiment, the invention relates tocompounds of formula IIb, where: Q is cyclohexyl, cycloheptyl, phenyl,benzodioxolyl, benzofuranyl, benzothiophenyl, furanyl, indolyl,pyrazolyl, pyridinyl, thiazolyl, or thiophenyl; Q is optionallysubstituted with 1-2 R⁸ groups independently selected from halo,—C₁₋₄alkyl, —C₀₋₄alkylene-OH, cyano, —C(O)O—C₁₋₄alkyl, —O—C₁₋₄alkyl,—S—C₁₋₄alkyl, and —CONH₂; and the alkyl groups in R⁸ are optionallysubstituted with 1 to 5 fluoro atoms. In another embodiment, theinvention relates to compounds of formula IIb, where Q is furanyl orthiophenyl.

Still another aspect of the invention relates to compounds havingformula IIc:

or a pharmaceutically acceptable salt thereof, where Z is as defined forformula I. In one particular embodiment, the invention relates tocompounds of formula IIc, where: Q is phenyl, furanyl, or thiophenyl;and the phenyl in Q is optionally substituted with 1-2 R⁸ groupsindependently selected from halo and —C₀₋₄alkylene-OH.

In yet another embodiment, the invention relates to a compound havingformula III:

or a pharmaceutically acceptable salt thereof, where R¹⁻³, R⁶⁻⁷, and Zare as defined for formula I. In one particular embodiment, theinvention relates to compounds of formula III where: R¹ is cyclopentyl;R² is phenyl; R³ is —OH; R⁶ and R⁷ are H; Z is —C₁₋₃alkylene-Q; Q isphenyl, benzofuranyl, furanyl, pyridinyl, or thiophenyl; and the phenylin Q is optionally substituted with 1-2 R⁸ groups independently selectedfrom halo and —C₀₋₄alkylene-OH.

In still another embodiment, the invention relates to a compound havingformula IV:

or a pharmaceutically acceptable salt thereof, where R¹⁻³, R⁶⁻⁷, and Zare as defined for formula I. In one particular embodiment, theinvention relates to compounds of formula IV where: R¹ is isobutyl orcyclopentyl; R² is phenyl; R³ is —OH; R⁶ and R⁷ are H; Z is —C₁₋₆alkylor —C₁₋₃alkylene-Q; Q is phenyl, furanyl, pyridinyl, or thiophenyl; Q isoptionally substituted with 1-2 R⁸ groups independently selected fromhalo, —C₁₋₄alkyl, —C₀₋₄alkylene-OH, and —O—C₁₋₄alkyl; and the alkylgroups in R⁵ are optionally substituted with 1 to 5 fluoro atoms.

A particular group of compounds of formula I are those disclosed in U.S.Provisional Application No. 60/967,914, filed on Sep. 7, 2007. Thisgroup includes compounds of formula (I′):

wherein: R¹ is selected from —C₁₋₆alkyl, —C₂₋₆alkenyl, and—C₃₋₉cycloalkyl; R² is aryl; R³ is selected from H and —C₀₋₁alkylene-OH;or forms a double bond with R¹; or —CR¹R²R³ together form a group offormula:

where A is a bond, —O—, —S—, —CH₂—, —CH═CH—, —CH₂CH₂—, —NH—, or—N(CH₃)—; and where R⁴ is selected from H, halo, —OH, —C₁₋₈alkyl, and—C₁₋₈alkoxy; X is a bond, —O— or —O—CH₂—; when X is a bond, Y is —CH₂—,Y′ is —N—, and Y″ is —CH₂—; when X is —O— or —O—CH₂—, Y′ is —CH—, and Yis a bond and Y″ is —CH₂— or —(CH₂)₂—, or Y is —CH₂— and Y″ is —CH₂—; R⁵is selected from fluoro and —C₁₋₄alkyl; and a is 0 or an integer of from1 to 3; R⁶ and R⁷ are independently selected from H and —C₁₋₄alkyl, andfurther wherein one of R⁶ or R⁷ may be —NH₂; Z is selected from H,—C₁₋₆alkyl, —C₁₋₃alkylene-Q, and —NH—C₀₋₁alkylene-Q, where Q is selectedfrom —C₃₋₇cycloalkyl, aryl, or heteroaryl, optionally substituted with1-5 R⁸ groups independently selected from halo, —C₁₋₄alkyl,—C₀₋₄alkylene-OH, cyano, —C₀₋₂alkylene-COOH, —C(O)O—C₁₋₄alkyl,—O—C₁₋₄alkyl, —S—C₁₋₄alkyl, —CONR^(8a)R^(8b), —NH—C(O)—C₁₋₄alkyl,—N-di-C₁₋₄alkyl, and —N⁺(O)O, where R^(8a) and R^(8b) are independentlyselected from H and —C₁₋₄alkyl; wherein R¹ and R² are optionallysubstituted with 1 to 5 R^(a) groups selected from —C₁₋₄alkyl,—C₂₋₄alkenyl, —C₂₋₄alkynyl, —C₃₋₆cycloalkyl, cyano, halo, —OR^(b),—C(O)OR^(b), —SR^(b), —S(O)R^(b), —S(O)₂R^(b), —C(O)NR^(c)R^(d) and—NR^(c)R^(d); where each R^(b) is independently selected from H,—C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl, and —C₃₋₆cycloalkyl; and eachR^(c) and R^(d) is independently selected from H, —C₁₋₄alkyl,—C₂₋₄alkenyl, —C₂₋₄alkynyl, and —C₃₋₆cycloalkyl; wherein each alkyl,alkenyl, alkynyl, alkylene, and cycloalkyl group in R^(a-d), R⁴⁻⁸, andZ, is optionally substituted with 1 to 5 fluoro atoms; wherein eachcycloalkyl in R^(a-d) is optionally substituted with 1 to 3 substituentsindependently selected from —C₁₋₄alkyl, —C₂₋₄alkenyl, —C₂₋₄alkynyl,cyano, halo, —O(C₁₋₄alkyl), —S(C₁₋₄alkyl), —S(O)(C₁₋₄alkyl),—S(O)₂(C₁₋₄alkyl), —NH₂, —NH(C₁₋₄alkyl) and —N(C₁₋₄alkyl)₂, wherein eachalkyl, alkenyl and alkynyl group is optionally substituted with 1 to 5fluoro substituents; and the alkylene group in Z is optionallysubstituted with 1 or 2 substituents independently selected from—C₁₋₂alkyl and —OH; or a pharmaceutically acceptable salt thereof.

In addition, particular compounds of the invention that are of interestinclude those set forth in the Examples below, as well as thepharmaceutically acceptable salts thereof.

Definitions

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

The term “alkyl” means a monovalent saturated hydrocarbon group whichmay be linear or branched. Unless otherwise defined, such alkyl groupstypically contain from 1 to 10 carbon atoms and include, for example—C₁₋₂alkyl, —C₁₋₄alkyl, and —C₁₋₆alkyl. Representative alkyl groupsinclude, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl,sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl and the like.

When a specific number of carbon atoms is intended for a particular termused herein, the number of carbon atoms is shown preceding the term assubscript. For example, the term “—C₁₋₄alkyl” means an alkyl grouphaving from 1 to 4 carbon atoms, and the term “—C₅₋₉cycloalkyl” means acycloalkyl group having from 5 to 9 carbon atoms, where the carbon atomsare in any acceptable configuration.

The term “alkylene” means a divalent saturated hydrocarbon group thatmay be linear or branched. Unless otherwise defined, such alkylenegroups typically contain from 0 to 10 carbon atoms and include, forexample, —C₀₋₁alkylene-, —C₀₋₂alkylene-, —C₀₋₄alkylene-, —C₀₋₅alkylene-,—C₁₋₄alkylene-, —C₁₋₂alkylene-, —C₂₋₄alkylene-, —C₂₋₅alkylene-, and—C₃₋₆alkylene-. Representative alkylene groups include, by way ofexample, methylene, ethane-1,2-diyl (“ethylene”), propane-1,2-diyl,propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl and the like. It isunderstood that when the alkylene term includes zero carbons such as—C₀₋₁alkylene- or —C₀₋₅alkylene-, such terms are intended to include theabsence of carbon atoms, that is, the alkylene group is not presentexcept for a covalent bond attaching the groups separated by thealkylene term.

The term “alkenyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon double bonds. Unless otherwisedefined, such alkenyl groups typically contain from 2 to 10 carbon atomsand include, for example, —C₂₋₄alkenyl and —C₂₋₆alkenyl. Representativealkenyl groups include, by way of example, ethenyl, n-propenyl,isopropenyl, n-but-2-enyl, n-hex-3-enyl and the like. The term“alkenylene” means a divalent alkenyl group, and exemplary alkenylenegroups include —C₂₋₃alkenylene-.

The term “alkoxy” means a monovalent group of the formula —O-alkyl,where alkyl is as defined herein. Unless otherwise defined, suchalkylene groups typically contain from 1 to 10 carbon atoms and include,for example, —C₁₋₄alkoxy and —C₁₋₈alkoxy. Representative alkoxy groupsinclude, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy,n-butoxy, sec-butoxy, isobutoxy, tert-butoxy and the like.

The term “alkynyl” means a monovalent unsaturated hydrocarbon groupwhich may be linear or branched and which has at least one, andtypically 1, 2 or 3, carbon-carbon triple bonds. Unless otherwisedefined, such alkynyl groups typically contain from 2 to 10 carbon atomsand include, for example, —C₂₋₄alkynyl and —C₂₋₆alkynyl. Representativealkynyl groups include, by way of example, ethynyl, n-propynyl,n-but-2-ynyl, n-hex-3-ynyl and the like.

The term “aryl” means a monovalent aromatic hydrocarbon having a singlering (i.e., phenyl) or fused rings (i.e., naphthalene). Unless otherwisedefined, such aryl groups typically contain from 6 to 10 carbon ringatoms and include, for example, —C₆₋₁₀aryl. Representative aryl groupsinclude, by way of example, phenyl and naphthalene-1-yl,naphthalene-2-yl, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclichydrocarbon group. Unless otherwise defined, such cycloalkyl groupstypically contain from 3 to 10 carbon atoms and include, for example,—C₃₋₆cycloalkyl, —C₃₋₇cycloalkyl, and —C₅₋₉cycloalkyl. Representativecycloalkyl groups include, by way of example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like.

The term “divalent hydrocarbon group” means a divalent hydrocarbon groupwhich is composed primarily of carbon and hydrogen atoms and whichoptionally contains one or more heteroatoms. Such divalent hydrocarbongroups may be branched or unbranched, saturated or unsaturated, acyclicor cyclic, aliphatic or aromatic, or combinations thereof. The divalenthydrocarbon group can optionally contain heteroatoms incorporated intothe hydrocarbon chain or as substituents attached to the hydrocarbonchain.

The term “halo” means fluoro, chloro, bromo and iodo.

As used herein, the phrase “having the formula” or “having thestructure” is not intended to be limiting and is used in the same waythat the term “comprising” is commonly used.

The term “heteroaryl” means a monovalent aromatic group having a singlering or two fused rings and containing in the ring at least oneheteroatom (typically 1 to 3 heteroatoms) selected from nitrogen, oxygenor sulfur. Unless otherwise defined, such heteroaryl groups typicallycontain from 5 to 10 total ring atoms and include, for example,—C₂₋₉heteroaryl. Representative heteroaryl groups include, by way ofexample, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, furanyl, thiophenyl,triazolyl, pyrazolyl, isoxazolyl, isothiazolyl, pyridinyl, pyrazinyl,pyridazinyl, pyrimidinyl, triazinyl, indolyl, benzofuranyl,benzopyranyl, benzothiophenyl, benzoimidazolyl, benzothiazolyl,benzodioxolyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl and thelike, where the point of attachment is at any available carbon ornitrogen ring atom.

The term “optionally substituted” means that group in question may beunsubstituted or it may be substituted one or several times, such as 1to 3 times or 1 to 5 times. For example, an alkyl group that is“optionally substituted” with 1 to 5 fluoro atoms, may be unsubstituted,or it may contain 1, 2, 3, 4, or 5 fluoro atoms.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug Administration.

The term “pharmaceutically acceptable salt” means a salt prepared from abase or an acid which is acceptable for administration to a patient,such as a mammal (e.g., salts having acceptable mammalian safety for agiven dosage regime). However, it is understood that the salts coveredby the invention are not required to be pharmaceutically acceptablesalts, such as salts of intermediate compounds that are not intended foradministration to a patient. Pharmaceutically acceptable salts can bederived from pharmaceutically acceptable inorganic or organic bases andfrom pharmaceutically acceptable inorganic or organic acids. Inaddition, when a compound of formula I contains both a basic moiety andan acidic moiety, zwitterions may be formed and are included within theterm “salt” as used herein. Salts derived from pharmaceuticallyacceptable inorganic bases include ammonium, calcium, copper, ferric,ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, andzinc salts, and the like. Salts derived from pharmaceutically acceptableorganic bases include salts of primary, secondary and tertiary amines,including substituted amines, cyclic amines, naturally-occurring aminesand the like, such as arginine, betaine, caffeine, choline,N,N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, isopropylamine, lysine, methylglucamine, morpholine,piperazine, piperadine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine, tromethamineand the like. Salts derived from pharmaceutically acceptable inorganicacids include salts of boric, carbonic, hydrohalic (hydrobromic,hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamicand sulfuric acids. Salts derived from pharmaceutically acceptableorganic acids include salts of aliphatic hydroxyl acids (e.g., citric,gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids),aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionicand trifluoroacetic acids), amino acids (e.g., aspartic and glutamicacids), aromatic carboxylic acids (e.g., benzoic, p-chlorobenzoic,diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatichydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic,1-hydroxy-naphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylicacids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic andsuccinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic,pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic,edisylic, ethanesulfonic, isethionic, methanesulfonic,naphthalenesulfonic, naphthalene-1,5-disulfonic,naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid,and the like.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment,i.e., the amount of drug needed to obtain the desired therapeuticeffect. For example, a therapeutically effective amount for treatingchronic obstructive pulmonary disease (COPD) is an amount of compoundneeded to, for example, reduce, suppress, eliminate or prevent, thesymptoms of (COPD), or to treat the underlying cause of (COPD). On theother hand, an “effective” amount is that amount needed to obtain adesired result, which may not necessarily be a therapeutically effectiveamount. For example, when studying a system comprising for antagonizinga muscarinic receptor, an “effective amount” may be the amount needed toantagonize the receptor.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD) in a patient,such as a mammal (particularly a human) that includes: (a) preventingthe disease or medical condition from occurring, i.e., prophylactictreatment of a patient; (b) ameliorating the disease or medicalcondition, i.e., eliminating or causing regression of the disease ormedical condition in a patient; (c) suppressing the disease or medicalcondition, i.e., slowing or arresting the development of the disease ormedical condition in a patient; or (d) alleviating the symptoms of thedisease or medical condition in a patient. For example, the term“treating COPD” would include preventing COPD from occurring,ameliorating COPD, suppressing COPD, and alleviating the symptoms ofCOPD. The term “patient” is intended to include those animals, such ashumans, that are in need of treatment or disease prevention, that arepresently being treated for disease prevention or treatment of aspecific disease or medical condition, as well as test subjects in whichcompounds of the invention are being evaluated or being used in a assay,for example an animal model.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

General Synthetic Procedures

Compounds of the invention can be prepared from readily availablestarting materials using the following general methods, the proceduresset forth in the Examples, or by using other methods, reagents, andstarting materials that are known to those of ordinary skill in the art.Although the following procedures may illustrate a particular embodimentof the invention, it is understood that other embodiments of theinvention can be similarly prepared using the same or similar methods orby using other methods, reagents and starting materials known to thoseof ordinary skill in the art. It will also be appreciated that wheretypical or preferred process conditions (i.e., reaction temperatures,times, mole ratios of reactants, solvents, pressures, etc.) are given,other process conditions can also be used unless otherwise stated. Whileoptimum reaction conditions will typically vary depending on variousreaction parameters such as the particular reactants, solvents andquantities used, those of ordinary skill in the art can readilydetermine suitable reaction conditions using routine optimizationprocedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary or desired to preventcertain functional groups from undergoing undesired reactions. Thechoice of a suitable protecting group for a particular functional groupas well as suitable conditions and reagents for protection anddeprotection of such functional groups are well-known in the art.Functional groups that may be protected so as to prevent undesiredreactions include, by way of example, carboxy groups, amino groups,hydroxyl groups, thiol groups, carbonyl groups and the like.Representative carboxy-protecting groups include, but are not limitedto, esters, such as methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl(PMB), 9-fluoroenylmethyl (Fm), trimethylsilyl (TMS),t-butyldimethylsilyl (TBS), diphenylmethyl (benzhydryl, DPM) and thelike; amides and hydrazides. Representative hydroxyl-protecting groupsinclude, but are not limited to, silyl groups includingtriC₁₋₆alkylsilyl groups, such as trimethylsilyl (TMS), triethylsilyl(TES), tert-butyldimethylsilyl (TBS) and the like; esters (acyl groups)including C₁₋₆alkanoyl groups, such as formyl, acetyl and the like;arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl (PMB),9-fluorenylmethyl (Fm), diphenylmethyl (benzhydryl, DPM) and the like;and ethers. Representative protecting groups for thiol groups includethioethers and thioesters. Representative protecting groups for carbonylgroups include acetals and ketals. Protecting groups other than thosedescribed herein may be used, if desired. For example, numerousprotecting groups, and their introduction and removal, are described inT. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis,Third Edition, Wiley, New York, 1999, and references cited therein. Morespecifically, the following abbreviations and reagents are used in theschemes presented below:

P represents an “amino-protecting group,” a term that is used herein tomean a protecting group suitable for preventing undesired reactions atan amino group. Representative amino-protecting groups include, but arenot limited to, t-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl(Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS),t-butyldimethylsilyl (TBDMS), and the like. Standard deprotectiontechniques are used to remove the P¹ group. For example, deprotection ofthe N-BOC group can use a reagent such as HCl or 4M HCl in 1,4-dioxane.

Suitable bases for use in these schemes include, by way of illustrationand not limitation, potassium carbonate, calcium carbonate, sodiumcarbonate, triethylamine, pyridine, 1,8-diazabicyclo-[5.4.0]undec-7-ene(DBU), N,N-diisopropylethylamine (DIPEA), sodium hydroxide, potassiumhydroxide, potassium t-butoxide, and metal hydrides.

Suitable inert diluents or solvents for use in these schemes include, byway of illustration and not limitation, tetrahydrofuran (THF),acetonitrile (MeCN), toluene, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), chloroform, carbontetrachloride (CHCl₃), 1,4-dioxane, methanol, ethanol, water, and thelike.

Suitable carboxylic acid/amine coupling reagents include1-hydroxybenzotriazole hydrate (HOBt),benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(PyBOP), O-(7-azabenzotriazol-1-yl-N,N,N′,N′ tetramethyluroniumhexafluorophosphate (HATU), dicyclohexylcarbodiimide (DCC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI),carbonyldiimidazole (CDT), and the like. Coupling reactions areconducted in an inert diluent in the presence of a base, and areperformed under conventional amide bond-forming conditions.

All reactions are typically conducted at a temperature within the rangeof about −78° C. to 100° C., for example at room temperature. Typically,reactions are monitored by use of thin layer chromatography (TLC), highperformance liquid chromatography (HPLC), and/or LCMS until completion.Reactions may be complete in minutes, or may take hours, typically from1-2 hours and up to 48 hours. Upon completion, the mixture may befurther treated in order to obtain the desired product. For example, themixture may be subjected to one or more of the following procedures:stripping or partitioning (e.g., between ethyl acetate and water orbetween 5% THF in ethyl acetate and 1M phosphoric acid); extraction(e.g., with ethyl acetate, CHCl₃, DCM, KOH/chloroform); washing (e.g.,with saturated aqueous NaCl, saturated NaHCO₃, Na₂CO₃ (5%), CHCl₃, HClor NaOH); drying (e.g., over MgSO₄ or Na SO₄); solvent removal (e.g., invacuo); filtering; being concentrated (e.g., in vacuo); and/orpurification (e.g., silica gel chromatography, flash chromatography, orreverse phase-HPLC).

By way of illustration, compounds of formula I can be prepared by one ormore of the following exemplary processes. The reactants are allcommercially available and/or can be readily synthesized by techniquesthat are well known in the art.

Formation of the Head Group where X is a Bond

Compound (3) is formed by coupling compounds (1) and (2) underconventional amide bond-forming conditions, followed by a deprotectionstep.

Examples of compound (1) include (R)-cyclopentylhydroxyphenyl aceticacid (R¹ is cyclopentyl, R² is phenyl and R³ is hydroxy). Examples ofcompound (2) include t-butyl 1-piperazinecarboxylate (a is 0, P¹ isBOC).

Formation of the Head Group where X is —O—

Compound (5) is formed by a Mitsunobu coupling reaction (Mitsunobu andYamada (1967) M. Bull. Chem. Soc. JPN. 40:2380-2382). Compound (1) andcompound (4) are reacted in the presence of a phosphine catalyst such astriphenylphospine and an azodicarboxylate such as diethylazodicarboxylate or diisopropyl azodicarboxylate, followed by adeprotection step to yield compound (5). Compound (5) can also beprepared by transesterification.

Examples of compound (1) include (R)-cyclopentylhydroxyphenyl aceticacid (R¹ is cyclopentyl, R² is phenyl and R³ is hydroxy). Examples ofcompound (4) include (R)-3-hydroxypyrrolidine-1-carboxylic acid t-butylester (a is 0, P¹ is BOC, Y is a bond, Y′ is —CH— and Y″ is —CH₂—),4-hydroxypiperidine-1-carboxylic acid t-butyl ester (a is 0, P¹ is BOC,Y is —CH₂—, Y′ is —CH—, and Y″ is —CH₂—) or(R)-3-hydroxypiperidine-1-carboxylic acid t-butyl ester (a is 0, P¹ isBOC, Y is a bond, Y′ is —CH— and Y″ is —(CH₂)₂—.

Addition of the Guanidine Moiety to the Head Group—Displacement of1^(st) Benzotriazole Moiety

DIPEA is added to compound (3) or compound (5) in an appropriatesolvent. Compound (6), the guanidinylating agent, is then added and thereaction stirred at room temperature until completion, typically from 30minutes to several hours, to yield compound (7), which is used directlyin the next step. Compound (6) is readily prepared by the methoddescribed in Katritzky et al. (2000) J. Org. Chem. 65(23):8080-8082. Anexample of compound (6) is C-(bis-benzotriazol-1-yl)methylene amine (R⁶is H).

Substituted Guanidine Formation Formation of Tri- or Tetra-substitutedGuanidine

Compound (7) is added to compound (8) and the mixture is maintained atroom temperature or heated (˜60° C.) until completion, typically about14-24 hours. The reaction is then cooled to room temperature, if needed,and the solvent removed. The crude material is then purified to afford acompound of formula I. Examples of compound (8) include2-thiophenemethyl amine, 4-hydroxybenzyl amine, and benzylamine.

Further details regarding specific reaction conditions and otherprocedures for preparing representative compounds of the invention orintermediates thereof are described in the Examples set forth below.

Utility

Compounds of the invention possess muscarinic receptor antagonistactivity, and in one embodiment, at nanomolar potencies. In oneembodiment, compounds of the invention are selective for inhibition ofM₃ muscarinic receptor subtype activity over M₂ muscarinic receptorsubtype activity. In another embodiment, compounds of the invention areselective for inhibition of M₃ and M₂ muscarinic receptor subtypeactivity over M₁, M₄, and M₅ muscarinic receptor subtype activity.Additionally, compounds of the invention are expected to possess adesirable duration of action. Accordingly, in another specificembodiment, the invention relates to compounds having a duration ofaction greater than about 24 hours. Moreover, compounds of the inventionare also expected to possess reduced side effects, such as dry mouth, atefficacious doses when administered by inhalation compared to otherknown muscarinic receptor antagonists administered by inhalation (suchas tiotropium).

One measure of the affinity of a compound for the M₃ receptor subtype isthe inhibition dissociation constant (K_(i)) for binding to thereceptor. Compounds of the invention are expected to have a K_(i) forthe M₃ receptor subtype of less than or equal to 100 nM, as determined,for example, by an in vitro radioligand displacement assay. Compounds ofparticular interest include those having a K_(i) less than or equal to50 nM, and in another embodiment, the compounds have a K_(i) less thanor equal to 10 nM, and in yet another embodiment, the compounds have aK_(i) less than or equal to 1.0 nM. Compounds of even more particularinterest include those having a K, less than or equal to 500 pM, and inanother embodiment, the compounds have a K_(i) less than or equal to 200pM. It is noted that in some cases, compounds of the invention maypossess weak muscarinic receptor antagonist activity. In such cases,those of skill in the art will recognize that these compounds still haveutility as research tools.

Also of particular interest are those compounds having an ID₅₀ of lessthan or equal to 100 μg/mL at 24 hours post dosing, more particularlythose compounds having an ID₅₀ of less than or equal to 30 μg/mL at 24hours post dosing.

Exemplary assays to determine properties of compounds of the invention,such as the muscarinic receptor antagonizing activity, are described inthe Examples and include by way of illustration and not limitation,assays that measure hM₁, hM₂, hM₃, hM₄, and hM₅ muscarinic receptorbinding (for example, as described in Assay 1). Useful functional assaysto determine the muscarinic receptor antagonizing activity of compoundsof the invention include by way of illustration and not limitation,assays that measure ligand-mediated changes in intracellular cyclicadenosine monophosphate (cAMP), ligand-mediated changes in activity ofthe enzyme adenylyl cyclase (which synthesizes cAMP), ligand-mediatedchanges in incorporation of guanosine 5′-O-(γ-thio)triphosphate([³⁵S]GTPγS) into isolated membranes via receptor catalyzed exchange of[³⁵S]GTPγS for GDP, ligand-mediated changes in free intracellularcalcium ions (measured, for example, with a fluorescence-linked imagingplate reader or FLIPR® from Molecular Devices, Inc.), and the like.Exemplary assays are described in Assay 2. Compounds of this inventionare expected to antagonize or decrease the activation of muscarinicreceptors in any of the assays listed above, or assays of a similarnature, and will typically be used in these studies at a concentrationranging from about 0.1-100 nanomolar. Thus, the aforementioned assaysare useful in determining the therapeutic utility, for example, thebronchodilating activity, of compounds of the invention.

Other properties and utilities of compounds of the invention can bedemonstrated using various in vitro and in vivo assays well-known tothose skilled in the art. For example, the in vivo potency of compoundsof the invention can be measured in an animal model such as theEinthoven model. Briefly, the bronchodilator activity of a compound isevaluated in an anesthetized animal (the Einthoven model), which usesventilation pressure as a surrogate measure of airway resistance. See,for example, Einthoven (1892) Pfugers Arch. 51:367-445; and Mohammed etal. (2000) Pulm Pharmacol Ther. 13(6):287-92, as well as Assay 3 whichdescribes a rat Einthoven model. In one embodiment, a compound of theinvention administered at a dose of 100 μg/ml in the rat Einthoven modelexhibits greater than or equal to 35% inhibition of thebronchoconstrictor response at 24 hours, and in another embodimentexhibits greater than or equal to 70% inhibition at 24 hours. Anotheruseful in vivo assay is the rat antisialagogue assay (for example, asdescribed in Assay 4).

Compounds of the invention are expected to be useful as therapeuticagents for treating medical conditions mediated by muscarinic receptors.Thus it is expected that patients suffering from a disease or disorderthat is treated by blocking the muscarinic receptor can be treated byadministering a therapeutically effective amount of a muscarinicreceptor antagonist of the invention. Such medical conditions include,by way of example, pulmonary disorders or diseases including thoseassociated with reversible airway obstruction, such as chronicobstructive pulmonary disease (e.g., chronic and wheezy bronchitis andemphysema), asthma, pulmonary fibrosis, allergic rhinitis, rhinorrhea,and the like. Other medical conditions that can be treated withmuscarinic receptor antagonists are genitourinary tract disorders, suchas overactive bladder or detrusor hyperactivity and their symptoms;gastrointestinal tract disorders, such as irritable bowel syndrome,diverticular disease, achalasia, gastrointestinal hypermotilitydisorders and diarrhea; cardiac arrhythmias, such as sinus bradycardia;Parkinson's disease; cognitive disorders, such as Alzheimer's disease;dismenorrhea; and the like.

The amount of active agent administered per dose or the total amountadministered per day may be predetermined or it may be determined on anindividual patient basis by taking into consideration numerous factors,including the nature and severity of the patient's condition, thecondition being treated, the age, weight, and general health of thepatient, the tolerance of the patient to the active agent, the route ofadministration, pharmacological considerations such as the activity,efficacy, pharmacokinetics and toxicology profiles of the active agentand any secondary agents being administered, and the like. Treatment ofa patient suffering from a disease or medical condition (such as COPD)can begin with a predetermined dosage or a dosage determined by thetreating physician, and will continue for a period of time necessary toprevent, ameliorate, suppress, or alleviate the symptoms of the diseaseor medical condition. Patients undergoing such treatment will typicallybe monitored on a routine basis to determine the effectiveness oftherapy. For example, in treating COPD, significant improvement inforced expiratory volume (measured in one second) may be used todetermine the effectiveness of treatment. Similar indicators for theother diseases and conditions described herein, are well-known to thoseskilled in the art, and are readily available to the treating physician.Continuous monitoring by the physician will insure that the optimalamount of active agent will be administered at any given time, as wellas facilitating the determination of the duration of treatment. This isof particular value when secondary agents are also being administered,as their selection, dosage, and duration of therapy may also requireadjustment. In this way, the treatment regimen and dosing schedule canbe adjusted over the course of therapy so that the lowest amount ofactive agent that exhibits the desired effectiveness is administeredand, further, that administration is continued only so long as isnecessary to successfully treat the disease or medical condition.

Accordingly, in one embodiment, compounds of the invention are usefulfor treating smooth muscle disorders in mammals, including humans andtheir companion animals (e.g., dogs, cats etc.). Such smooth muscledisorders include, by way of illustration, overactive bladder, chronicobstructive pulmonary disease and irritable bowel syndrome. Typically,suitable doses for treating smooth muscle disorders or other disordersmediated by muscarinic receptors will range from about 0.14 μg/kg/day toabout 7 mg/kg/day of active agent; including from about 0.15 μg/kg/dayto about 5 mg/kg/day. For an average 70 kg human, this would amount toabout 10 μg per day to about 500 mg per day of active agent.

In a specific embodiment, compounds of the invention are useful fortreating pulmonary or respiratory disorders, such as COPD or asthma, inmammals including humans, by administering to a patient atherapeutically effective amount of the compound. Generally, the dosefor treating a pulmonary disorder will range from about 10-1500 μg/day.The term “COPD” is understood by those of ordinary skill in the art toinclude a variety of respiratory conditions, including chronicobstructive bronchitis and emphysema, as exemplified by the teachings ofBarnes (2000) N. Engl. J. Med. 343:269-78, and references cited therein.When used to treat a pulmonary disorder, compounds of the invention areoptionally administered in combination with other therapeutic agentssuch as a β₂-adrenoreceptor agonist; a corticosteroid, a non-steroidalanti-inflammatory agent, or combinations thereof.

When administered by inhalation, compounds of the invention typicallyhave the effect of producing bronchodilation. Accordingly, in another ofits method aspects, the invention relates to a method of producingbronchodilation in a patient, comprising administering to a patient abronchodilation-producing amount of a compound of the invention.Generally, the therapeutically effective dose for producingbronchodilation will range from about 10-1500 μg/day.

In another embodiment, compounds of the invention are used to treatoveractive bladder. When used to treat overactive bladder, a typicaldose will range from about 1.0-500 mg/day. In yet another embodiment,compounds of the invention are used to treat irritable bowel syndrome.When used to treat irritable bowel syndrome, compounds of the inventionwill typically be administered orally or rectally, and a typical dosewill range from about 1.0-500 mg/day.

Since compounds of this invention possess muscarinic receptor antagonistactivity, such compounds are also useful as research tools forinvestigating or studying biological systems or samples havingmuscarinic receptors. Any suitable biological system or sample havingM₁, M₂, M₃, M₄ and/or M₅ muscarinic receptors may be employed in suchstudies which may be conducted either in vitro or in vivo.Representative biological systems or samples suitable for such studiesinclude, but are not limited to, cells, cellular extracts, plasmamembranes, tissue samples, isolated organs, mammals (such as mice, rats,guinea pigs, rabbits, dogs, pigs, humans, and so forth), and the like,with mammals being of particular interest. In one particular embodimentof the invention a muscarinic receptor in a mammal is antagonized byadministering a muscarinic receptor-antagonizing amount of a compound ofthe invention. Compounds of the invention can also be used as researchtools by conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprising amuscarinic receptor is typically contacted with a muscarinicreceptor-antagonizing amount of a compound of the invention. After thebiological system or sample is exposed to the compound, the effects ofantagonizing the muscarinic receptor are determined using conventionalprocedures and equipment, such as by measuring binding in a radioligandbinding assays or ligand-mediated changes in a functional assay or bydetermining the amount of bronchoprotection provided by the compound ina bronchoprotection assay in a mammal. Exposure encompasses contactingcells or tissue with the compound, administering the compound to amammal, for example by i.p. or i.v. administration, and so forth. Thisdetermining step may comprise measuring a response, i.e., a quantitativeanalysis or may comprise an observation, i.e., a qualitative analysis.Measuring a response involves, for example, determining the effects ofthe compound on the biological system or sample using conventionalprocedures and equipment, such as radioligand binding assays andmeasuring ligand-mediated changes in functional assays. The assayresults can be used to determine the activity level as well as theamount of compound necessary to achieve the desired result, i.e., amuscarinic-antagonizing amount. Typically, the determining step willinvolve determining the muscarinic receptor ligand-mediated effects.

Additionally, compounds of the invention can be used as research toolsfor evaluating other chemical compounds, and thus are also useful inscreening assays to discover, for example, new compounds havingmuscarinic receptor binding activity. In this manner, a compound of theinvention is used as a standard in an assay to allow comparison of theresults obtained with a test compound and with compounds of theinvention to identify those test compounds that have about equal orsuperior binding, if any. For example, muscarinic receptor binding data(as determined, for example, by in vitro radioligand displacementassays) for a test compound or a group of test compounds is compared tothe muscarinic receptor binding data for a compound of the invention toidentify those test compounds that have the desired properties, e.g.,test compounds having binding about equal or superior to a compound ofthe invention, if any. Alternatively, for example, bronchoprotectiveeffects can be determined for test compounds and a compound of theinvention in a bronchoprotection assay in a mammal and this datacompared to identify test compounds providing about equal or superiorbronchoprotective effects. This aspect of the invention includes, asseparate embodiments, both the generation of comparison data (using theappropriate assays) and the analysis of the test data to identify testcompounds of interest. Thus, a test compound can be evaluating in abiological assay, by a method comprising the steps of: (a) conducting abiological assay with a test compound to provide a first assay value;(b) conducting the biological assay with a compound of the invention toprovide a second assay value; wherein step (a) is conducted eitherbefore, after or concurrently with step (b); and (c) comparing the firstassay value from step (a) with the second assay value from step (b).Exemplary biological assays include muscarinic receptor binding assays.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient inthe form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to,inhaled, oral, nasal, topical (including transdermal) and parenteralmodes of administration. Further, the compounds of the invention may beadministered, for example orally, in multiple doses per day, in a singledaily dose or a single weekly dose. It will be understood that any formof the compounds of the invention, (i.e., free base, pharmaceuticallyacceptable salt, solvate, etc.) that is suitable for the particular modeof administration can be used in the pharmaceutical compositionsdiscussed herein.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of the invention. The compositions may containother therapeutic and/or formulating agents if desired. A “compound ofthe invention” may also be referred to herein as the “active agent.”

The pharmaceutical compositions of this invention typically contain atherapeutically effective amount of a compound of the invention. Thoseskilled in the art will recognize, however, that a pharmaceuticalcomposition may contain more than a therapeutically effective amount,i.e., bulk compositions, or less than a therapeutically effectiveamount, i.e., individual unit doses designed for multiple administrationto achieve a therapeutically effective amount. In one embodiment, thecomposition will contain from about 0.01-95 wt % of active agent,including, from about 0.01-30 wt %, such as from about 0.01-10 wt %,with the actual amount depending upon the formulation itself, the routeof administration, the frequency of dosing, and so forth. In anotherembodiment, a composition suitable for inhalation, for example,comprises from about 0.01-30 wt % or active agent with yet anotherembodiment comprises from about 0.01-10 wt % active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers and the like usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable forinhaled administration. Suitable compositions for inhaled administrationwill typically be in the form of an aerosol or a powder. Suchcompositions are generally administered using well-known deliverydevices, such as a nebulizer inhaler, a dry powder inhaler, or ametered-dose inhaler, examples of which are described below.

In a specific embodiment of the invention, a composition comprising theactive agent is administered by inhalation using a nebulizer inhaler.Such nebulizer devices typically produce a stream of high velocity airthat causes the composition to spray as a mist that is carried into apatient's respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the active agent is typically dissolved in a suitablecarrier to form a solution. Alternatively, the active agent can bemicronized and combined with a suitable carrier to form a suspension ofmicronized particles of respirable size, where micronized is typicallydefined as having particles in which at least about 90 percent of theparticles have a mass median diameter of less than about 10 μm. The term“mass median diameter” means the diameter such that half the mass of theparticles is contained in particles with larger diameter and half iscontained in particles with smaller diameter.

Suitable nebulizer devices include the Respimat® Soft Mist™ Inhaler(Boehringer Ingelheim), the AERx® Pulmonary Delivery System (AradigmCorp.), and the PARI LC Plus Reusable Nebulizer (Pari GmbH). Anexemplary composition for use in a nebulizer inhaler comprises anisotonic aqueous solution comprising from about 0.05 μg/mL to about 10mg/mL of a compound of the invention. In one embodiment, such a solutionhas a pH of about 4-6.

In another specific embodiment of the invention, a compositioncomprising the active agent is administered by inhalation using a drypowder inhaler (DPI). Such DPIs typically administer the active agent asa free-flowing powder that is dispersed in a patient's air-stream duringinspiration. In order to achieve a free flowing powder, the active agentis typically formulated with a suitable excipient such as lactose,starch, mannitol, dextrose, polylactic acid, polylactide-co-glycolide,and combinations thereof. Typically, the active agent is micronized andcombined with an excipient to form a blend suitable for inhalation.Accordingly, in one embodiment of the invention, the active agent is inmicronized form. For example, a representative composition for use in aDPI comprises dry lactose having a particle size between about 1 μm andabout 100 μm (e.g., dry milled lactose) and micronized particles of theactive agent. Such a dry powder formulation can be made, for example, bycombining lactose with the active agent and then dry blending thecomponents. Alternatively, if desired, the active agent can beformulated without an excipient. The composition is then typicallyloaded into a DPI, or into inhalation cartridges or capsules for usewith a DPI. DPIs are well known to those of ordinary skill in the art,and many such devices are commercially available, with representativedevices including Aerolizer® (Novartis), Airmax™ (WAX), ClickHaler®(Innovata Biomed), Diskhaler® (GlaxoSmithKline), Diskus® or Accuhaler(GlaxoSmithKline), Easyhaler® (Orion Pharma), Eclipse™ (Aventis),FlowCaps® (Hovione), Handihaler® (Boehringer Ingelheim), Pulvinal®(Chiesi), Rotahaler® (GlaxoSmithKline), SkyeHaler™ or Certihaler™(SkyePharma), Twisthaler (Schering-Plough), Turbuhaler® (AstraZeneca),Ultrahaler® (Aventis), and the like.

In yet another specific embodiment of the invention, the compositioncomprising the active agent is administered by inhalation using ametered-dose inhaler (MDI). Such MDIs typically discharge a measuredamount of the active agent using compressed propellant gas. Metered-doseformulations thus typically comprise a solution or suspension of theactive agent in a liquefied propellant, such as a chlorofluorocarbonsuch as CCl₃F or a hydrofluoroalkane (HFA) such as1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane (HFA 227), although HFAs aregenerally preferred due to concerns about chlorofluorocarbons affectingthe ozone layer. Additional optional components of HFA formulationsinclude co-solvents, such as ethanol or pentane, and surfactants, suchas sorbitan trioleate, oleic acid, lecithin, and glycerin. See, forexample, U.S. Pat. No. 5,225,183 to Purewal et al., EP 0717987 A2(Minnesota Mining and Manufacturing Company), and WO 92/22286 (MinnesotaMining and Manufacturing Company). A representative composition for usein an MDI comprises from about 0.01-5 wt % of active agent; from about0-20 wt % ethanol; and from about 0-5 wt % surfactant; with theremainder being an HFA propellant. Such compositions are typicallyprepared by adding a chilled or pressurized hydrofluoroalkane to asuitable container containing the active agent, ethanol (if present) andthe surfactant (if present). To prepare a suspension, the active agentis micronized and then combined with the propellant. The formulation isthen loaded into an aerosol canister, which forms a portion of the MDI.MDIs are well known to those of ordinary skill in the art, and many suchdevices are commercially available, with representative devicesincluding AeroBid Inhaler System (Forest Pharmaceuticals), AtroventInhalation Aerosol (Boehringer Ingelheim), Flovent® (GlaxoSmithKline),Maxair Inhaler (3M), Proventil® Inhaler (Schering), Serevent® InhalationAerosol (GlaxoSmithKline), and the like. Alternatively, a suspensionformulation can be prepared by spray drying a coating of surfactant onmicronized particles of the active agent. See, for example, WO 99/53901(Glaxo Group Ltd.) and WO 00/61108 (Glaxo Group Ltd.).

Additional examples of processes of preparing respirable particles, andformulations and devices suitable for inhalation dosing are described inU.S. Pat. No. 5,874,063 to Briggner et al.; U.S. Pat. No. 5,983,956 toTrofast; U.S. Pat. No. 6,221,398 to Jakupovic et al.; U.S. Pat. No.6,268,533 to Gao et al.; U.S. Pat. No. 6,475,524 to Bisrat et al.; andU.S. Pat. No. 6,613,307 to Cooper.

In another embodiment, the pharmaceutical compositions are suitable fororal administration. Suitable compositions for oral administration maybe in the form of capsules, tablets, pills, lozenges, cachets, dragees,powders, granules; solutions or suspensions in an aqueous or non-aqueousliquid; oil-in-water or water-in-oil liquid emulsions; elixirs orsyrups; and the like; each containing a predetermined amount of theactive agent.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the composition will typicallycomprise the active agent and one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate. Solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as cetyl alcohol and/or glycerol monostearate; absorbents,such as kaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate sodiumsulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, if appropriate, with oneor more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Suspensions maycontain suspending agents such as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, i.e., each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

Compounds of the invention can also be administered parenterally (e.g.,by subcutaneous, intravenous, intramuscular, or intraperitonealinjection). For such administration, the active agent is provided in asterile solution, suspension, or emulsion. Exemplary solvents forpreparing such formulations include water, saline, low molecular weightalcohols such as propylene glycol, polyethylene glycol, oils, gelatin,fatty acid esters such as ethyl oleate, and the like. A typicalparenteral formulation is a sterile pH 4-7 aqueous solution of theactive agent. Parenteral formulations may also contain one or moresolubilizers, stabilizers, preservatives, wetting agents, emulsifiers,and dispersing agents. These formulations may be rendered sterile by useof a sterile injectable medium, a sterilizing agent, filtration,irradiation, or heat.

Compounds of the invention can also be administered transdermally usingknown transdermal delivery systems and excipients. For example, thecompound can be admixed with permeation enhancers, such as propyleneglycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and thelike, and incorporated into a patch or similar delivery system.Additional excipients including gelling agents, emulsifiers and buffers,may be used in such transdermal compositions if desired.

If desired, the compounds of this invention may be administered incombination with one or more other therapeutic agents. Thus, in oneembodiment, compositions of the invention may optionally contain otherdrugs that are co-administered with a compound of the invention. Forexample, the composition may further comprise one or more drugs (alsoreferred to as “secondary agents(s)”) selected from the group of otherbronchodilators (e.g., PDE₃ inhibitors, adenosine 2b modulators and β₂adrenergic receptor agonists); anti-inflammatory agents (e.g., steroidalanti-inflammatory agents such as corticosteroids and glucocorticoids;non-steroidal anti-inflammatory agents (NSAIDs); and PDE₄ inhibitors);other muscarinic receptor antagonists (i.e., antichlolinergic agents);antiinfective agents (e.g., Gram positive and Gram negative antibiotics,and antiviral agents); antihistamines; protease inhibitors; afferentblockers (e.g., D₂ agonists and neurokinin modulators); and combinationsthereof. Numerous examples of such therapeutic agents are well known inthe art, and examples are described below. By combining a compound ofthe invention with a secondary agent, double therapy can be achieved,i.e., muscarinic receptor antagonist activity and activity associatedwith the secondary agent (e.g., β₁ adrenergic receptor agonist), in somecases by administering two compositions and in some cases byadministering a single composition containing the active agent and thesecondary agent. Accordingly, in yet another aspect of the invention, apharmaceutical composition comprises a compound of the invention, asecond active agent, and a pharmaceutically acceptable carrier. Third,fourth etc. active agents may also be included in the composition. Forexample, a composition may comprise a compound of the invention; asecondary agent selected from corticosteroids, β₂ adrenergic receptoragonists; phosphodiesterase-4 inhibitors, and combinations thereof; anda pharmaceutically acceptable carrier. In a specific embodiment, thecomposition comprises a compound of the invention, a β₂ adrenergicreceptor agonist, and a steroidal anti-inflammatory agent. Incombination therapy, the amount of compound of the invention that isadministered, as well as the amount of secondary agents, may be lessthan the amount typically administered in monotherapy.

A compound of the invention may be either physically mixed with thesecond active agent to form a composition containing both agents; oreach agent may be present in separate and distinct compositions whichare administered to the patient simultaneously or sequentially. Forexample, a compound of the invention can be combined with a secondactive agent using conventional procedures and equipment to form acombination of active agents comprising a compound of the invention anda second active agent. Additionally, the active agents may be combinedwith a pharmaceutically acceptable carrier to form a pharmaceuticalcomposition comprising a compound of the invention, a second activeagent and a pharmaceutically acceptable carrier. In this embodiment, thecomponents of the composition are typically mixed or blended to create aphysical mixture. The physical mixture is then administered in atherapeutically effective amount using any of the routes describedherein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention. In other embodiments this timed relationshipis less than 12 hours, less than 8 hours, less than 6 hours, less than 4hours, less than 3 hours, less than 1 hour, less than thirty minutes,less than ten minutes, less than one minute, or immediately afteradministration of the compound of the invention. This is also referredto as sequential administration. Thus, a compound of the invention canbe administered by inhalation simultaneously or sequentially withanother active agent using an inhalation delivery device that employsseparate compartments (e.g. blister packs) for each active agent, wheresequential may mean being administered immediately after administrationof the compound of the invention or at some predetermined time later(e.g., one hour later or three hours later). Alternatively, thecombination may be administered using separate delivery devices, i.e.,one delivery device for each agent. Additionally, the agents can bedelivered by different routes of administration, i.e., one by inhalationand the other by oral administration.

In one embodiment, the kit comprises a first dosage form comprising acompound of the invention and at least one additional dosage formcomprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc,) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount. i.e., are typically administered in an amount thatproduces a therapeutically beneficial effect when co-administered with acompound of the invention. The secondary agent can be in the form of apharmaceutically acceptable salt, solvate, optically pure stereoisomer,and so forth. Thus, secondary agents listed below are intended toinclude all such forms, and are commercially available or can beprepared using conventional procedures and reagents. Suitable doses fora secondary agent are typically in the range of about 0.05 μg/day toabout 500 mg/day.

In a particular embodiment, a compound of the invention is administeredin combination with a β₂ adrenergic receptor agonist. Representative β₂adrenergic receptor agonists include, but are not limited to, albuterol,bitolterol, fenoterol, formoterol, indacaterol, isoetharine,levalbuterol, metaproterenol, pirbuterol, salbutamol, salmefamol,salmeterol, terbutaline, and the like. Other β₂ adrenergic receptoragonists that can be used in combination with compounds of the inventioninclude, but are not limited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)-phenyl]ethyl}amino)-hexyl]oxy}-butyl)benzenesulfonamideand3-(-3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}-amino)heptyl]oxy}-propyl)benzenesulfonamideand related compounds disclosed in WO 02/066422 (Glaxo Group Ltd.);3-[3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]oxy}butyl)phenyl]imidazolidine-2,4-dioneand related compounds disclosed in WO 02/070490 (Glaxo Group Ltd.);3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)-hexyl]oxy}butyl)benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxy-phenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide,N-(t-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide,N-(t-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide,N-(t-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide and related compounds disclosed in WO02/076933 (Glaxo Group Ltd.);4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)-oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds disclosed in WO 03/024439 (Glaxo Group Ltd.);N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)-ethylamineand related compounds disclosed in U.S. Pat. No. 6,576,793 to Moran etal.;N-{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,653,323 to Moran etal. In a particular embodiment, the β₂-adrenoreceptor agonist is acrystalline monohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)-phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamine.Typically, the β₂-adrenoreceptor agonist will be administered in anamount sufficient to provide from about 0.05-500 μg per dose.

In a particular embodiment, a compound of the invention is administeredin combination with a steroidal anti-inflammatory agent. Representativesteroidal anti-inflammatory agents include, but are not limited to,beclomethasone dipropionate; budesonide; butixocort propionate;20R-16α,17α-[butylidenebis(oxy)]-6α,9α-difluoro-11β-hydroxy-17β-(methylthio)androsta-4-en-3-one(RPR-106541); ciclesonide; dexamethasone;6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester;6α,9α-difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester;6α,9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxyandrosta-1,4-diene-17β-carbothioicacid (S)-(2-oxotetrahydrofuran-3S-yl) ester; flunisolide; fluticasonepropionate; methyl prednisolone; mometasone furoate; prednisolone;prednisone; rofleponide; ST-126; triamcinolone acetonide; and the like.Typically, the steroidal anti-inflammatory agent will be administered inan amount sufficient to provide from about 0.05-500 μg per dose.

An exemplary combination is a compound of the invention co-administeredwith salmeterol as the β₂ adrenergic receptor agonist, and fluticasonepropionate as the steroidal anti-inflammatory agent. Another exemplarycombination is a compound of the invention co-administered with acrystalline monohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)-ethylamineas the β₂-adrenoreceptor agonist, and6α,9α-difluoro-17α-[(2-furanylcarbonyl)-oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester as the steroidal anti-inflammatory agent.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (such as sodium cromoglycate;nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g.,theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g., monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors, such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g., adenosine 2a agonists); cytokineantagonists (e.g., chemokine antagonists such as, an interleukinantibody (αIL antibody), specifically, an αIL-4 therapy, an αIL-13therapy, or a combination thereof); or inhibitors of cytokine synthesis.

In a particular embodiment, a compound of the invention is administeredin combination with a phosphodiesterase-4 (PDE4) inhibitors or mixedPDE3/PDE4 inhibitors. Representative PDE4 or mixed PDE3/PDE4 inhibitorsinclude, but are not limited to, c is4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxy-phenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-cyclohexane-1-carboxylicacid and the like, or pharmaceutically acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds disclosed in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofylline(Almirall-Prodesfarma); VM554/UM565 (Vernalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

In a particular embodiment, a compound of the invention is administeredin combination with a muscarinic antagonist (i.e., anticholinergicagent). Representative muscarinic antagonists include, but are notlimited to, atropine, atropine sulfate, atropine oxide, methylatropinenitrate, homatropine hydrobromide, hyoscyamine (d, l) hydrobromide,scopolamine hydrobromide, ipratropium bromide, oxitropium bromide,tiotropium bromide, methantheline, propantheline bromide, anisotropinemethyl bromide, clidinium bromide, copyrrolate (Robinul), isopropamideiodide, mepenzolate bromide, tridihexethyl chloride (Pathilone),hexocyclium methylsulfate, cyclopentolate hydrochloride, tropicamide,trihexyphenidyl hydrochloride, pirenzepine, telenzepine, AF-DX 116 andmethoctramine and the like.

In a particular embodiment, a compound of the invention is administeredin combination with an antihistamine (i.e., H₁-receptor antagonist).Representative antihistamines include, but are not limited to,ethanolamines, such as carbinoxamine maleate, clemastine fumarate,diphenylhydramine hydrochloride and dimenhydrinate; ethylenediamines,such as pyrilamine amleate, tripelennamine hydrochloride andtripelennamine citrate; alkylamines, such as chlorpheniramine andacrivastine; piperazines, such as hydroxyzine hydrochloride, hydroxyzinepamoate, cyclizine hydrochloride, cyclizine lactate, meclizinehydrochloride and cetirizine hydrochloride; piperidines, such asastemizole, levocabastine hydrochloride, loratadine or itsdescarboethoxy analogue, terfenadine and fexofenadine hydrochloride;azelastine hydrochloride; and the like.

The following formulations illustrate representative pharmaceuticalcompositions of the invention.

Exemplary Compositions for Administration by a DPI

A compound of the invention (0.2 mg) is micronized and then blended withlactose (25 mg). This blended mixture is then loaded into a gelatininhalation cartridge. The contents of the cartridge are administeredusing a DPI, for example.

A micronized compound of the invention (100 mg) is blended with milledlactose (25 g) (e.g., lactose in which not greater than about 85% of theparticles have a MMD of about 60 μm to about 90 μm and not less than 15%of the particles have a MMD of less then 15 μm). This blended mixture isthen loaded into individual blisters of a peelable blister pack in anamount sufficient to provide about 10 μg to about 500 μg of the compoundof the invention per dose. The contents of the blisters are administeredusing a DPI.

Alternately, a micronized compound of the invention (1 g) is blendedwith milled lactose (200 g) to form a bulk composition having a weightratio of compound to milled lactose of 1:200. The blended composition ispacked into a DPI capable of delivering between about 10 μg to about 500μg of the compound of the invention per dose.

Alternately, a micronized compound of the invention (100 mg) and amicronized β₂ adrenergic receptor agonist (500 mg) are blended withmilled lactose (30 g). The blended mixture is then loaded intoindividual blisters of a peelable blister pack in an amount sufficientto provide about 10 μg to about 500 μg of the compound of the inventionper dose. The contents of the blisters are administered using a DPI.

Exemplary Compositions for Use in an MDI

A micronized compound of the invention (10 g) is dispersed in a solutionprepared by dissolving lecithin (0.2 g) in demineralized water (200 mL).The resulting suspension is spray dried and then micronized to form amicronized composition comprising particles having a mean diameter lessthan about 1.5 μm. The micronized composition is then loaded into MDIcartridges containing pressurized 1,1,1,2-tetrafluoroethane in an amountsufficient to provide about 10 μg to about 500 μg of the compound of theinvention per dose when administered by the MDI.

Alternately, a suspension containing 5 wt % compound of the invention,0.5 wt % lecithin, and 0.5 wt % trehalose is prepared by dispersing 5 gof a compound of the invention as micronized particles with mean sizeless than 10 μm in a colloidal solution formed from 0.5 g of trehaloseand 0.5 g of lecithin dissolved in 100 mL of demineralized water. Thesuspension is spray dried and the resulting material is micronized toparticles having a mean diameter less than 1.5 μm. The particles areloaded into canisters with pressurized 1,1,1,2-tetrafluoroethane.

Exemplary Composition for Use in a Nebulizer Inhaler

A compound of the invention (25 mg) is dissolved in citrate buffered (pH5) isotonic saline (125 mL). The mixture is stirred and sonicated untilthe compound is dissolved. The pH of the solution is checked andadjusted, if necessary, to pH 5 by slowly adding aqueous 1N sodiumhydroxide. The solution is administered using a nebulizer device thatprovides about 10 μg to about 500 μg of the compound of the inventionper dose.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), spray-dried lactose (440 g) andmagnesium stearate (10 g) are thoroughly blended. The resultingcomposition is then loaded into hard gelatin capsules (500 mg ofcomposition per capsule).

Exemplary Suspension for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of compound per 10 mL of suspension:

Ingredients Amount Compound of the invention  1.0 g Fumaric acid  0.5 gSodium chloride  2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 gGranulated sugar 25.5 g Sorbitol (70% solution) 12.85 g  Veegum ® K(magnesium aluminum silicate)  1.0 g Flavoring 0.035 mL Colorings 0.5 mgDistilled water q.s. to 100 mL

Exemplary Injectable Formulation for Administration by Injection

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetatebuffer solution (2.0 mL). The pH of the resulting solution is adjustedto pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodiumhydroxide, as necessary, and then sufficient water for injection isadded to provide a total volume of 20 mL. The mixture is then filteredthrough a sterile filter (0.22 micron) to provide a sterile solutionsuitable for administration by injection.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard meaning:

AC adenylyl cyclase

BSA bovine serum albumin

cAMP 3′-5′ cyclic adenosine monophosphate

CHO Chinese hamster ovary

cM₅ cloned chimpanzee M₅ receptor

DCM dichloromethane (i.e., methylene chloride)

DIPEA N,N-diisopropylethylamine

dPBS Dulbecco's phosphate buffered saline

DMF N,N-dimethylformamide

EDCI N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

EDTA ethylenediamine tetraacetic acid

EtOAc ethyl acetate

FBS fetal bovine serum

FLIPR fluorometric imaging plate reader

HBSS Hank's Buffered Salt Solution

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

hM₁ cloned human M₁ receptor

hM₂ cloned human M₂ receptor

hM₃ cloned human M₃ receptor

hM₄ cloned human M₄ receptor

hM₅ cloned human M₅ receptor

HOBt 1-hydroxybenzotriazole hydrate

MCh methylcholine

MeOH methanol

TFA trifluoroacetic acid

THF tetrahydrofuran

Any other abbreviations used herein but not defined have their standard,generally accepted meaning. Unless noted otherwise, all materials, suchas reagents, starting materials and solvents, were purchased fromcommercial suppliers (such as Sigma-Aldrich, Fluka Riedel-de Haën, andthe like) and were used without further purification. Reactions were rununder nitrogen atmosphere, unless noted otherwise. Progress of reactionmixtures was monitored by thin layer chromatography (TLC), analyticalhigh performance liquid chromatography (anal. HPLC), and massspectrometry, the details of which are given below and separately inspecific examples of reactions. Reaction mixtures were worked up asdescribed specifically in each reaction; commonly they were purified byextraction and other purification methods such as temperature-, andsolvent-dependent crystallization, and precipitation. In addition,reaction mixtures were routinely purified by preparative HPLC.

Preparation 1 (R)-cyclopentylhydroxyphenyl Acetic Acid

(2R,5R)-2-t-Butyl-5-phenyl-1,3-dioxolan-4-one (1a): (R)-Mandelic acid(20 g, 130 mmol) was dissolved in anhydrous pentane (200 mL, 1.7 mol).Pivaldehyde (13.6 g, 153 mmol) was added followed bytrifluoromethanesulfonic acid (488 μL, 5.4 mmol). The reaction wasallowed to reflux at 36° C. under nitrogen. After 5.5 hours, the mixturewas allowed to cool to room temperature before stirring with 200 mL ofan 8 wt % NaHCO₃ solution for 10 minutes. Excess pentane was removed byrotary evaporation. The solids were collected by filtration and rinsed(100 mL water) while under vacuum filtration. The solids were driedovernight under high vacuum to yield intermediate (1a) as a white solid(23.8 g, 88% purity).

(2R,5S)-2-t-Butyl-5-(1-hydroxycyclopentyl)-5-phenyl-1,3-dioxolan-4-one(1b): Lithium hexamethyldisilazide (0.8 g, 4.7 mmol; 4.7 mL of 1.0 M inhexanes) was added to anhydrous THF (5.3 mL, 65 mmol) at −78° C.Intermediate (1a) (800 mg, 3.6 mmol) in 5.3 mL anhydrous THF was addedto the solution dropwise over 15 minutes. After 30 minutescyclopentanone (451 μL, 5.1 mmol) was added dropwise over less than 1minute. After 2 hours, 0.8 mL of saturated aqueous Na₂HPO₄ was added,and the mixture stirred at room temperature for 5 minutes. The mixturewas added to 8 mL saturated aqueous ammonium chloride. The aqueous layerwas washed (2×80 mL EtOAc), and the organic layers were combined, driedover Na₂SO₄, filtered and concentrated. The crude product (780 mg) waspurified by flash chromatography (5-15% EtOAc gradient over 30 minuteswith hexanes) to yield intermediate (1b).

(2R,5S)-2-t-Butyl-5-cyclopent-1-enyl-5-phenyl-1,3-dioxolan-4-one (1c):Intermediate (1b) (650 mg, 2.1 mmol) was dissolved in 6.8 mL anhydrousTHF and the solution was cooled to 0° C. Thionyl chloride (436 μL, 6mmol) was added dropwise, followed by the addition of pyridine (777 μL,9.6 mmol). The mixture was stirred at 0° C. for 1 hour. Saturatedaqueous ammonium chloride (14 mL) was added and the mixture was stirredfor 5 minutes while warming to room temperature. The layers wereseparated, and the aqueous layer was washed (2×100 mL EtOAc.). Theorganic layers were combined, dried over Na₂SO₄, filtered andconcentrated to yield intermediate (1c) as a light yellow oil (540 mg),which was used in the next step without further purification.

(S)-Cyclopent-1-enyl-hydroxyphenylacetic acid (1d): Intermediate (1c)(540 mg, 1.9 mmol) was dissolved in MeOH (927 μL, 22.9 mmol). Water(1.84 mL, 102 mmol) was added, followed by the addition of KOH (1.1 g,18.8 mmol). The reaction was refluxed at 130° C. for 3 hours. Themixture was diluted to 250 mL with saturated ammonium chloride, thenwashed (2×100 mL hexane). The remaining aqueous emulsion was washed(2×250 mL EtOAc). The EtOAc layers were combined, washed with 50 mLsaturated aqueous NaCl, dried over Na₂SO₄, filtered and concentrated toyield intermediate (1d) as a brownish-yellow solid (290 mg).

Intermediate (1d) (280 mg, 1.3 mmol) was dissolved in MeOH (2.50 mL,61.7 mmol) and the reaction flask was flushed with nitrogen before 28 mgof 10% Pd/C was added to the mixture. The mixture was stirred at roomtemperature under 1 atm hydrogen and the reaction was monitored by HPLCuntil the starting material was consumed (˜24 hours). The reactionvessel was flushed with nitrogen, then the mixture was filtered throughcelite and rinsed with MeOH. The filtrate was concentrated under vacuumto obtain the title compound as a slightly yellow solid (284 mg).

Preparation 2(R)-2-Cyclopentyl-2-hydroxy-2-phenyl-1-piperazin-1-ylethanone

To a stirred solution of (R)-cyclopentylhydroxyphenyl acetic acid (10.0g, 45.4 mmol) in DCM (200 mL) was added t-butyl 1-piperazinecarboxylate(8.5 g, 45.4 mmol). Into the reaction was added DIPEA (23.7 mL, 13.6mmol), HOBt (10.4 g, 68.1 mmol), and then EDCI (10.4 g, 54.5 mmol). Themixture was stirred at room temperature for 12 hours. The mixture wasthen washed with 1N NaOH (300 mL), 1N HCl (300 mL) then saturatedaqueous NaCl (300 mL). The organic layer was then removed, dried overMgSO₄ and then filtered. The solvent was removed under reduced pressure.A solution of 20% TFA/DCM was added to the crude material, and theresulting mixture was stirred for 2 hours at room temperature. Thesolvent was removed under reduced pressure. DCM (300 mL) was added andthe mixture was washed with saturated sodium bicarbonate (300 mL). Theorganic layer was then removed, dried over MgSO₄ and filtered. The crudematerial was purified via silica gel chromatography (10% MeOH/DCM w/1%NH₃ (aq)) to afford the title compound as a white powder (9.0 g, 31.2mmol).

Example 14-((R)-2-Cyclopentyl-2-hydroxy-2-phenylacetyl)-N-thiophen-2-ylmethylpiperazine-1-carboxamidine

To a stirred solution of(R)-2-cyclopentyl-2-hydroxy-2-phenyl-1-piperazin-1-ylethanone (3.9 g13.7 mmol) in DMF (200 mL) was added DIPEA (4.8 mL, 27.3 mmol), and thenC-(bis-benzotriazol-1-yl)methylene amine (3.6 g, 13.7 mmol). This wasstirred at room temperature for 30 minutes, followed by the addition ofC-thiophen-2-yl-methylamine (2.8 mL, 27.3 mmol). The mixture was heatedat 60° C. for 14 hours. The reaction was cooled to room temperature andthe solvent removed by reduced pressure. The crude material was purifiedby reverse phase-HPLC to afford the title compound as a TFA salt (0.7 g,1.3 mmol). MS m/z: [M+H]⁺calcd for C₂₃H₃₀N₄O₂S, 427.21; found 427.2.

Alternate Synthesis

DIPEA (7.3 mL, 41.6 mmol) was added to(R)-2-cyclopentyl-2-hydroxy-2-phenyl-1-piperazin-1-ylethanone (6.0 g,20.8 mmol) dissolved in ethanol (90 mL, 2 mol).C-(bis-benzotriazol-1-yl)-methyleneamine (6.0 g, 22.9 mmol) was addedand the mixture stirred at room temperature for 30 minutes.C-Thiophen-2-yl-methylamine (4.9 g, 41.6 mmol) was added and the mixturewas stirred overnight at 55° C. The mixture was condensed and theproduct purified by HPLC to afford the title compound as a TFA salt (7.3g, 98% purity). MS m/z: [M+H]⁺calcd for C₂₃H₃₀N₄O₂S, 427.21; found427.4.

Example 24-((R)-2-Cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(4-hydroxybenzyl)piperazine-1-carboxamidine

To a stirred solution of(R)-2-cyclopentyl-2-hydroxy-2-phenyl-1-piperazin-1-ylethanone (5.00 g17.3 mmol; prepared as described in Preparation 1) in DMF (200 mL) wasadded DIPEA (10.6 mL, 60.7 mmol) and thenC-(bis-benzotriazol-1-yl)-methylene amine (5.48 g, 20.8 mmol). This wasstirred at room temperature for 30 minutes, followed by the addition of4-hydroxybenzyl amine (12.0 g mL, 97 mmol). The mixture was heated at60° C. for 14 hours. The reaction was cooled to room temperature and thesolvent removed by reduced pressure. The crude material was purified byreverse phase-HPLC to afford the title compound as a TFA salt (1.7 g,3.1 mmol). MS m/z: [M+H]⁺calcd for C₂₅H₃₂N₄O₃, 437.25; found 437.2.

Example 34-((R)-2-Cyclopentyl-2-hydroxy-2-phenylacetyl)-N-furan-2-ylmethylpiperazine-1-carboxamidine

(R)-2-Cyclopentyl-2-hydroxy-2-phenyl-1-piperazin-1-ylethanone (2.0 g,6.9 mmol) and C-(bis-benzotriazol-1-yl)methyleneamine (2.0 g, 7.6 mmol)were added to ethanol (40.0 mL, 685 mmol), followed by the addition ofDIPEA (2.4 mL, 13.9 mmol). The resulting mixture was stirred at roomtemperature for about 1 hour until all solids were dissolved, to formthe intermediate. Furfurylamine (1.2 mL, 13.9 mmol) was added and thereaction mixture was stirred at 35° C. until the reaction was complete(about 22 hours). Purification by preparative HPLC yielded the titlecompound as a TFA salt (329 mg, 6.9 mmol, 97.5% purity). MS m/z:[M+H]⁺calcd for C₂₃H₃₀N₄O₃, 411.23; found 411.2.

Example 4

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds4-1 to 4-52, having the following, were also prepared as TFA salts:

Ex. Z Q 4-1 —CH(CH₃)—Q phenyl 4-2 —CH₂—Q 3,4-difluorophenyl 4-3 —CH₂—Q4-methoxyphenyl 4-4 —CH₂—Q thiophen-3-yl 4-5 —CH₂—Q phenyl 4-6 —CH₂—Q3-fluorophenyl 4-7 —(CH₂)₂—Q phenyl 4-8 —CH₂—Q pyridin-2-yl 4-9 —CH₂—Q3-hydroxyphenyl 4-10 —CH₂—Q 4-fluorophenyl 4-11 —CH₂—Q 2-fluorophenyl4-12 —CH₂—Q cyclohexyl 4-13 —CH₂—Q 3-methoxyphenyl 4-14 —CH₂—Q3,5-difluorophenyl 4-15 —CH₂—Q thiazol-2-yl 4-16 —CH₂—Q 1H-pyrazol-3-yl4-17 —NH—Q phenyl 4-18 —CH₂—Q furan-3-yl 4-19 —CH₂—Q2-methyl-thiazol-4-yl 4-20 —CH₂—Q propyl 4-21 —CH₂—Q butyl 4-22 —CH₂—Qpentyl 4-23 —NH—Q 2-fluorophenyl 4-24 —NH—Q 2-chlorophenyl 4-25 —NH—Q3-fluorophenyl 4-26 —NH—Q 3-chlorophenyl 4-27 —NH—Q 4-methylphenyl 4-28—NH—Q 4-fluorophenyl 4-29 —NH—Q 4-chlorophenyl 4-30 —NH—Q4-methoxyphenyl 4-31 —CH₂—Q 4-benzoic acid methyl ester 4-32 —CH₂—Q1H-indol-2-yl 4-33 —CH₂—Q cycloheptyl 4-34 —CH₂—Q 2-hydroxyphenyl 4-35—CH₂—Q 4-trifluoromethoxyphenyl 4-36 —CH₂—Q 4-amidophenyl 4-37 —CH₂—Q4-hydroxymethylphenyl 4-38 —CH₂—Q 1H-indol-5-yl 4-39 —CH₂—Qbenzofuran-5-yl 4-40 —CH₂—Q 4-methylphenyl 4-41 —CH₂—Q4-methylsulfanylphenyl 4-42 —CH₂—Q 3-cyanophenyl 4-43 —CH₂—Q3-amidophenyl 4-44 —CH₂—Q 2-methylphenyl 4-45 —CH₂—Q 3-methylphenyl 4-46—CH₂—Q 1H-indo1-4-yl 4-47 —CH₂—Q 3-methylsulfanylphenyl 4-48 —CH₂—Qbenzo[b]thiophen-5-yl 4-49 —CH₂—Q benzo[1,3]dioxo1-5-yl 4-50 —CH₂—Qbenzo[b]thiophen-2-yl 4-51 —CH₂—Q 1-methyl-1H-pyrazol-3-yl 4-52 —CH₂—Qcyclopentyl

-   (4-1)    4-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N—((R)-1-phenylethyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₄N₄O₂, 435.27; found 435.2.-   (4-2)    4-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(3,4-difluorobenzyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₅H₃₀F₂N₄O₂, 457.23; found 457.2.-   (4-3)    4-(2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(4-methoxybenzyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₄N₄O₃, 451.26; found 451.2.-   (4-4)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-thiophen-3-ylmethyl-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₃H₃₀N₄O₂S, 427.21; found 427.4.-   (4-5)    N-benzyl-4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₅H₃₂N₄O₂, 421.25; found 421.2.-   (4-6)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(3-fluorobenzyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₅H₃₁FN₄O₂, 439.24; found 439.2.-   (4-7)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-phenethylpiperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₄N₄O₂, 435.27; found 435.2.-   (4-8)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-pyridin-2-ylmethyl-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₄H₃₁N₅O₂, 422.25; found 422.2.-   (4-9)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(3-hydroxybenzyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₅H₃₂N₄O₃, 437.25; found 437.2.-   (4-10)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(4-fluorobenzyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₅H₃₁FN₄O₂, 439.24; found 439.2.-   (4-11)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(2-fluorobenzyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₅H₃₁FN₄O₂, 439.24; found 439.2.-   (4-12)    N-cyclohexylmethyl-4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₅H₃₈N₄O₂, 427.30; found 427.2.-   (4-13)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(3-methoxybenzyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₄N₄O₃, 451.26; found 451.2.-   (4-14)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(3,5-difluorobenzyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₅H₃₀F₂N₄O₂, 457.23; found 457.2.-   (4-15)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-thiazol-2-ylmethyl-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₂H₂₉N₅O₂S, 428.20; found 428.2.-   (4-16)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(1H-pyrazol-3-ylmethyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₂H₃₀N₆O₂, 411.24; found 411.2.-   (4-17)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(phenylamino)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₄H₃₁N₅O₂, 422.25; found 422.2.-   (4-18)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-furan-3-ylmethylpiperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₃H₃₀N₄O₃, 411.23; found 411.2.-   (4-19)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(2-methylthiazol-4-ylmethyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₃H₃₁N₅O₂S, 442.22; found 442.2.-   (4-20)    N-butyl-4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₂H₃₄N₄O₂, 387.27; found 387.2.-   (4-21)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-pentylpiperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₃H₃₆N₄O₂, 401.28; found 401.2.-   (4-22)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-hexylpiperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₄H₃₈N₄O₂, 415.30; found 415.2.-   (4-23)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(2-fluorophenyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₄H₃₀FN₅O₂, 440.24; found 440.2.-   (4-24)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(2-chlorophenyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₄H₃₀ClN₅O₂, 456.21; found 456.2.-   (4-25)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(3-fluorophenyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₄H₃₀FN₅O₂, 440.24; found 440.2.-   (4-26)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(3-chlorophenyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₄H₃₀ClN₅O₂, 456.21; found 456.2.-   (4-27)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(4-methylphenyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₅H₃₃N₅O₂, 436.26; found 436.2.-   (4-28)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(4-fluorophenyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₄H₃₀FN₅O₂, 440.24; found 440.2.-   (4-29)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(4-chlorophenyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₄H₃₀ClN₅O₂, 456.21; found 456.2.-   (4-30)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(4-methoxyphenyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₅H₃₃N₅O₃, 452.26; found 452.2.-   (4-31)    4-({[4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperazine-1-carboximidoyl]amino}methyl)benzoic    acid methyl ester. MS m/z: [M+H]⁺calcd for C₂₇H₃₄N₄O₄, 479.26; found    479.2.-   (4-32)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(1H-indol-2-ylmethyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₇H₃₃N₅O₂, 460.26; found 460.2.-   (4-33)    N-cycloheptylmethyl-4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₄₀N₄O₂, 441.32; found 441.2.-   (4-34)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(2-hydroxybenzyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₅H₃₂N₄O₃, 437.25; found 437.2.-   (4-35)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(4-trifluoromethoxybenzyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₁F₃N₄O₃, 505.24; found 505.2.-   (4-36)    4-({[4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperazine-1-carboximidoyl]amino}methyl)benzamide.    MS m/z: [M+H]⁺calcd for C₂₆H₃₃N₅O₃, 464.26; found 464.2.-   (4-37)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(4-hydroxymethylbenzyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₄N₄O₃, 451.26; found 451.2.-   (4-38)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(1H-indol-5-ylmethyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₇H₃₃N₅O₂, 460.26; found 460.2.-   (4-39)    N-benzofuran-5-ylmethyl-4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₇H₃₂N₄O₃, 461.25; found 461.2.-   (4-40)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(4-methylbenzyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₄N₄O₂, 435.27; found 435.2.-   (4-41)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(4-methylsulfanylbenzyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₄N₄O₂S, 467.24; found 467.2.-   (4-42)    N-(3-cyanobenzyl)-4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₁N₅O₂, 446.25; found 446.2.-   (4-43)    3-({[4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-piperazine-1-carboximidoyl]amino}methyl)benzamide.    MS m/z: [M+H]⁺calcd for C₂₆H₃₃N₅O₃, 464.26; found 464.2.-   (4-44)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(2-methylbenzyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₄N₄O₂, 435.27; found 434.2.-   (4-45)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(3-methylbenzyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₄N₄O₂, 435.27; found 435.2.-   (4-46)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(1H-indol-4-ylmethyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₇H₃₃N₅O₂, 460.26; found 460.2.-   (4-47)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(3-methylsulfanylbenzyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₄N₄O₂S, 467.24; found 467.2.-   (4-48)    N-benzo[b]thiophen-5-ylmethyl-4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₇H₃₂N₄O₂S, 477.22; found 477.2.-   (4-49)    N-benzo[1,3]dioxol-5-ylmethyl-4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₆H₃₂N₄O₄, 465.24; found 465.2.-   (4-50)    N-benzo[b]thiophen-2-ylmethyl-4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₇H₃₂N₄O₂S, 477.22; found 477.2.-   (3-51)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-(1-methyl-1H-pyrazol-3-ylmethyl)piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₃H₃₂N₆O₂, 425.26; found 425.2.-   (3-52)    4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-cyclopentylmethyl-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₄H₃₆N₄O₂, 413.28; found 413.2.

Example 5

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds5-1 and 5-2, having the following, were also prepared as TFA salts:

-   (5-1)    N-benzyl-4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N′-methyl-piperazine-1-carboxamidine    (R⁶=—CH₃). MS m/z: [M+H]⁺calcd for C₂₆H₃₄N₄O₂, 435.27; found 435.2.-   (5-2)    N-benzyl-4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N′-ethylpiperazine-1-carboxamidine    (R⁶=—CH₂CH₃). MS m/z: [M+H]⁺calcd for C₂₇H₃₆N₄O₂, 449.28; found    449.2.

Preparation 3R)-3-((R)-2-Cyclopentyl-2-hydroxy-2-phenylacetoxy)pyrrolidine-1-carboxylicacid t-butyl ester

At room temperature, diisopropyl azodicarboxylate (980 μl, 5 mmol) wasslowly added to a mixture of (R)-cyclopentylhydroxyphenylacetic acid(1.1 g, 5 mmol), (R)-3-hydroxypyrrolidine-1-carboxylic acid t-butylester (1.0 g, 5.5 mmol), and triphenyl-phosphine (1.3 g, 5 mmol) in 10ml of THF. The reaction mixture was then stirred at room temperatureovernight.

The solvent was removed and 100 ml of EtOAc was added. The organic layerwas washed with a sodium bicarbonate solution (50 ml×3), then saturatedaqueous NaCl, and dried over sodium sulfate. The solvent was removed,providing 4 g of crude product, which was purified by flashchromatography (EtOAc/hexane) to yield the title compound (1.5 g, 99%purity).

Preparation 4 (R)-Cyclopentylhydroxyphenylacetic acid(R)-pyrrolidin-3-yl ester

(R)-3-((R)-2-Cyclopentyl-2-hydroxy-2-phenylacetoxy)pyrrolidine-1-carboxylicacid t-butyl ester (1.3 g) in 8 ml 1,4-dioxane was added to 4 ml of 4MHCl in 1,4-dioxane and stirred at room temperature overnight to yieldthe title compound as an HCl salt (1 g).

Example 6 (R)-Cyclopentylhydroxyphenylacetic acid(R)-1-(N-benzylcarbamimidoyl)pyrrolidin-3-yl ester

(R)-3-(R)-2-Cyclopentyl-2-hydroxy-2-phenylacetoxy)pyrrolidine-1-carboxylicacid t-butyl ester (97.5 mg, 0.3 mmol) in 3 ml of DMF was added to DIPEA(130 μl, 750 μmol), followed by the addition ofC-(bis-benzotriazol-1-yl)-methyleneamine (87 mg, 330 μmol). The reactionmixture was stirred at room temperature for 2 hours, followed by theaddition of benzylamine (39 μL, 360 μmol). The reaction mixture was thestirred at room temperature overnight. The solvent was removed and thesolute purified by reverse phase chromatography to yield the titlecompound as a TFA salt (78.2 mg, 99.5% purity). MS m/z: [M+H]⁺calcd forC₂₅H₃₁N₃O₃, 422.24; found 422.2.

Example 7

Following the procedures described in the previous example, andsubstituting the appropriate starting materials and reagents, compounds7-1 to 7-18, having the following formula, were also prepared as TFAsalts:

Ex. Q 7-1 phenyl 7-2 thiophen-2-yl 7-3 2-hydroxyphenyl 7-43-hydroxyphenyl 7-5 4-hydroxyphenyl 7-6 furan-2-yl 7-7 furan-3-yl 7-82-fluorophenyl 7-9 3-fluorophenyl 7-10 4-fluorophenyl 7-112,6-difluorophenyl 7-12 3,4-difluorophenyl 7-13 3,5-difluorophenyl 7-144-trifluoromethylphenyl 7-15 5-methylfuran-2-yl 7-16 pyridin-2-yl 7-17—(CH₂)₂-phenyl 7-18 —CH₂-phenyl

-   (7-1) (R)-cyclopentylhydroxyphenylacetic acid    1-(N-benzylcarbamimidoyl)-pyrrolidin-3-yl ester. MS m/z: [M+H]⁺calcd    for C₂₅H₃₁N₃O₃, 422.24; found 422.4.-   (7-2) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-(N-thiophen-2-ylmethyl-carbamimidoyl)pyrrolidin-3-yl ester. MS    m/z: [M+H]⁺calcd for C₂₃H₂₉N₃O₃S, 428.19; found 428.4.-   (7-3) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-[N-(2-hydroxybenzyl)-carbamimidoyl]pyrrolidin-3-yl ester. MS    m/z: [M+H]⁺calcd for C₂₅H₃₁N₃O₄, 438.23; found 438.5.-   (7-4) (R)-cyclopentylhydroxyphenyl acetic acid    (R)-1-[N-(3-hydroxybenzyl)-carbamimidoyl]pyrrolidin-3-yl ester. MS    m/z: [M+H]⁺calcd for C₂₅H₃₁N₃O₄, 438.23; found 438.5.-   (7-5) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-[N-(4-hydroxybenzyl)-carbamimidoyl]pyrrolidin-3-yl ester. MS    m/z: [M+H]⁺calcd for C₂₅H₃₁N₃O₄, 438.23; found 438.3.-   (7-6) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-(N-furan-2-ylmethyl-carbamimidoyl)pyrrolidin-3-yl ester. MS    m/z: [M+H]⁺calcd for C₂₃H₂₉N₃O₄, 412.22; found 412.2.-   (7-7) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-(N-furan-3-ylmethyl-carbamimidoyl)pyrrolidin-3-yl ester. MS    m/z: [M+H]⁺calcd for C₂₃H₂₉N₃O₄, 412.22; found 412.2.-   (7-8) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-[N-(2-fluoro-benzyl)-carbamimidoyl]pyrrolidin-3-yl ester MS    m/z: [M+H]⁺calcd for C₂₅H₃₀FN₃O₃, 440.23; found 440.3.-   (7-9) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-[N-(3-fluoro-benzyl)-carbamimidoyl]pyrrolidin-3-yl ester. MS    m/z: [M+H]⁺calcd for C₂₅H₃₀FN₃O₃, 440.23; found 440.4.-   (7-10) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-[N-(4-fluorobenzyl)-carbamimidoyl]pyrrolidin-3-yl ester. MS    m/z: [M+H]⁺calcd for C₂₅H₃₀FN₃O₃, 440.23; found 440.4.-   (7-11) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-[N-(2,6-difluorobenzyl)-carbamimidoyl]pyrrolidin-3-yl ester.    MS m/z: [M+H]⁺calcd for C₂₅H₂₉F₂N₃O₃, 458.22; found 458.2.-   (7-12) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-[N-(3,4-difluorobenzyl)-carbamimidoyl]pyrrolidin-3-yl ester.    MS m/z: [M+H]⁺calcd for C₂₅H₂₉F₂N₃O₃, 458.22; found 458.2.-   (7-13) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-[N-(3,5-difluorobenzyl)-carbamimidoyl]pyrrolidin-3-yl ester.    MS m/z: [M+H]⁺calcd for C₂₅H₂₉F₂N₃O₃, 458.22; found 458.2.-   (7-14) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-[N-(4-trifluoromethoxybenzyl)-carbamimidoyl]pyrrolidin-3-yl    ester. MS m/z: [M+H]⁺calcd for C₂₆H₃₀F₃N₃O₄, 506.22; found 506.2.-   (7-15) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-[N-(5-methyl-furan-2-ylmethyl)carbamimidoyl]pyrrolidin-3-yl    ester. MS m/z: [M+H]⁺calcd for C₂₄H₃₁N₃O₄, 426.23; found 426.2.-   (7-16) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-(N-pyridin-2-ylmethyl-carbamimidoyl)pyrrolidin-3-yl ester. MS    m/z: [M+H]⁺calcd for C₂₄H₃₀N₄O₃, 423.23; found 423.2.-   (7-17) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-[N-(3-phenylpropyl)-carbamimidoyl]pyrrolidin-3-yl ester. MS    m/z: [M+H]⁺calcd for C₂₇H₃₅N₃O₃, 450.27; found 450.2.-   (7-18) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-(N-phenethylcarbamimidoyl)-pyrrolidin-3-yl ester. MS m/z:    [M+H]⁺calcd for C₂₆H₃₃N₃O₃, 436.25; found 436.2.

Example 8

Following the procedures described in the previous example, andsubstituting the appropriate starting materials and reagents, compounds8-1 to 8-13, having the following formula, were also prepared as TFAsalts:

Ex. Q 8-1 phenyl 8-2 thiophen-2-yl 8-3 furan-2-yl 8-4 furan-3-yl 8-52-hydroxyphenyl 8-6 3-hydroxyphenyl 8-7 4-hydroxyphenyl 8-83-methoxyphenyl 8-9 2-fluorophenyl 8-10 3-fluorophenyl 8-114-fluorophenyl 8-12 pyridin-2-yl 8-13 benzofuran-5-yl

-   (8-1) (R)-cyclopentylhydroxyphenylacetic acid    1-(N-benzylcarbamimidoyl)piperidin-4-yl ester. MS m/z: [M+H]⁺calcd    for C₂₆H₃₃N₃O₃, 436.25; found 436.5.-   (8-2) (R)-cyclopentylhydroxyphenylacetic acid    1-(N-thiophen-2-ylmethyl-carbamimidoyl)piperidin-4-yl ester. MS m/z:    [M+H]⁺calcd for C₂₄H₃₁N₃O₃S, 442.21; found 442.4.-   (8-3) (R)-cyclopentylhydroxyphenylacetic acid    1-(N-furan-2-ylmethyl-carbamimidoyl)piperidin-4-yl ester. MS m/z:    [M+H]⁺calcd for C₂₄H₃₁N₃O₄, 426.23; found 426.2.-   (8-4) (R)-cyclopentylhydroxyphenylacetic acid    1-(N-furan-3-ylmethyl-carbamimidoyl)piperidin-4-yl ester. MS m/z:    [M+H]⁺calcd for C₂₄H₃₁N₃O₄, 426.23; found 426.2.-   (8-5) (R)-cyclopentylhydroxyphenylacetic acid    1-[N-(2-hydroxybenzyl)-carbamimidoyl]piperidin-4-yl ester. MS m/z:    [M+H]⁺calcd for C₂₆H₃₃N₃O₄, 452.25; found 452.2.-   (8-6) (R)-cyclopentylhydroxyphenylacetic acid    1-[N-(3-hydroxybenzyl)-carbamimidoyl]piperidin-4-yl ester. MS m/z:    [M+H]⁺calcd for C₂₆H₃₃N₃O₄, 452.25; found 452.2.-   (8-7) (R)-cyclopentylhydroxyphenylacetic acid    1[N-(4-hydroxybenzyl)-carbamimidoyl]piperidin-4-yl ester. MS m/z:    [M+H]⁺calcd for C₂₆H₃₃N₃O₄, 452.25; found 452.2.-   (8-8) (R)-cyclopentylhydroxyphenylacetic acid    1-[N-(3-methoxybenzyl)-carbamimidoyl]piperidin-4-yl ester. MS m/z:    [M+H]⁺calcd for C₂₇H₃₅N₃O₄, 466.26; found 466.2.-   (8-9) (R)-cyclopentylhydroxyphenylacetic acid    1-[N-(2-fluorobenzyl)-carbamimidoyl]piperidin-4-yl ester. MS m/z:    [M+H]⁺calcd for C₂₆H₃₂FN₃O₃, 454.24; found 454.2.-   (8-10) (R)-cyclopentylhydroxyphenylacetic acid    1-[N-(3-fluorobenzyl)-carbamimidoyl]piperidin-4-yl ester. MS m/z:    [M+H]⁺calcd for C₂₆H₃₂FN₃O₃, 454.24; found 454.2.-   (8-11) (R)-cyclopentylhydroxyphenylacetic acid    1-[N-(4-fluorobenzyl)-carbamimidoyl]piperidin-4-yl ester. MS m/z:    [M+H]⁺calcd for C₂₆H₃₂FN₃O₃, 454.24; found 454.2.-   (8-12) (R)-cyclopentylhydroxyphenylacetic acid    1-(N-pyridin-2-ylmethyl-carbamimidoyl)piperidin-4-yl ester. MS m/z:    [M+H]⁺calcd for C₂₅H₃₂N₄O₃, 437.25; found 437.2.-   (8-13) (R)-cyclopentylhydroxyphenylacetic acid    1-(N-benzofuran-5-ylmethyl-carbamimidoyl)piperidin-4-yl ester. MS    m/z: [M+H]⁺calcd for C₂₈H₃₃N₃O₄, 476.25; found 476.4.

Example 9

Following the procedures described in the previous example, andsubstituting the appropriate starting materials and reagents,(R)-cyclopentylhydroxyphenylacetic acid(R)-1-(N-benzylcarbamimidoyl)piperidin-3-yl ester was also prepared as aTFA salt:

MS m/z: [M+H]⁺calcd for C₂₆H₃₃N₃O₃, 436.25; found 436.5.

Example 10

Following the procedures described in the previous example, andsubstituting the appropriate starting materials and reagents,(R)-cyclopentylhydroxyphenylacetic acid1-[N-(3-phenylpropyl)carbamimidoyl]piperidin-4-yl ester was alsoprepared as a TFA salt:

MS m/z: [M+H]⁺calcd for C₂₈H₃₇N₃O₃, 464.28; found 464.4.

Example 11

Following the procedures described in the previous example, andsubstituting the appropriate starting materials and reagents, compounds11-1 and 11-2, having the following formula, were also prepared as TFAsalts:

-   (11-1) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-(N-methylcarbamimidoyl)-pyrrolidin-3-yl ester (Z=—CH₃). MS    m/z: [M+H]⁺calcd for C₁₉H₂₇N₃O₃, 346.21; found 346.2.-   (11-2) (R)-cyclopentylhydroxyphenylacetic acid    (R)-1-(N-butylcarbamimidoyl)-pyrrolidin-3-yl ester (Z=—(CH₂)₃CH₃).    MS m/z: [M+H]⁺calcd for C₂₂H₃₃N₃O₃, 388.25; found 388.2.

Example 12

Following the procedures described in the previous example, andsubstituting the appropriate starting materials and reagents,(R)-cyclopentylhydroxyphenylacetic acid 1-carbamimidoyl-piperidin-4-ylester was also prepared as a TFA salt:

MS m/z: [M+H]⁺calcd for C₁₉H₂₇N₃O₃, 346.21; found 346.1.

Example 13

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds13-1 to 13-2, having the following formula, were also prepared as TFAsalts:

-   (13-1) 2-hydroxy-4-methyl-2-phenylpentanoic acid    (R)-1-(N-benzylcarbamimidoyl)-pyrrolidin-3-yl ester (Q=phenyl). MS    m/z: [M+H]⁺calcd for C₂₄H₃₁N₃O₃, 410.24; found 410.2.-   (13-2) 2-hydroxy-4-methyl-2-phenylpentanoic acid    (R)-1[N-(4-hydroxybenzyl)-carbamimidoyl]pyrrolidin-3-yl ester    (Q=4-hydroxyphenyl). MS m/z: [M+H]⁺calcd for C₂₄H₃₁N₃O₄, 426.23;    found 426.2.

Example 14

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds14-1 to 14-4, having the following formula, were also prepared as TFAsalts:

Ex. Q 14-1 phenyl 14-2 thiophen-2-yl 14-3 4-hydroxyphenyl 14-4furan-2-yl

-   (14-1)    N-benzyl-4-(2-hydroxy-2,2-di-thiophen-2-ylacetyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₂H₂₄N₄O₂S₂, 441.13; found 441.0.-   (14-2)    4-(2-hydroxy-2,2-di-thiophen-2-ylacetyl)-N-thiophen-2-ylmethyl-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₀H₂₂N₄O₂S₃, 447.09; found 447.0.-   (14-3)    N-(4-hydroxybenzyl)-4-(2-hydroxy-2,2-di-thiophen-2-yl-acetyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₂H₂₄N₄O₃S₂, 457.13; found 457.0.-   (14-4)    N-furan-2-ylmethyl-4-(2-hydroxy-2,2-di-thiophen-2-yl-acetyl)-piperazine-1-carboxamidine.    MS m/z: [M+H]⁺calcd for C₂₀H₂₂N₄O₃S₂, 431.11; found 431.0.

Example 15

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents, compounds15-1 to 15-2, having the following formula, were also prepared as TFAsalts:

-   (15-1) 9H-xanthene-9-carboxylic acid    1[N-(3-phenylpropyl)carbamimidoyl]piperidin-4-yl ester    (Z=—(CH₂)₃-phenyl). MS m/z: [M+H]⁺calcd for C₂₉H₃₁N₃O₃, 470.24;    found 470.4.-   (15-2) 9H-xanthene-9-carboxylic acid    (R)-1-(N-benzylcarbarnimidoyl)pyrrolidin-3-yl ester (Z=—CH₂-phenyl).    MS m/z: [M+H]⁺calcd for C₂₆H₂₅N₃O₃, 428.19; found 428.5.

Example 16

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents,9H-xanthene-9-carboxylic acid (S)-1-carbamimidoylpiperidin-3-ylmethylester was also prepared as a TFA salt:

MS m/z: [M+H]⁺calcd for C₂₁H₂₃N₃O₃, 366.17; found 366.2.

Example 17

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents,9H-xanthene-9-carboxylic acid (R)-1-carbamimidoylpyrrolidin-3-ylmethylester was also prepared as a TFA salt:

MS m/z: [M+H]⁺calcd for C₂₀H₂₁N₃O₃, 352.16; found 352.2.

Example 18

Following the procedures described in the previous examples, andsubstituting the appropriate starting materials and reagents,9H-xanthene-9-carboxylic acid 1-carbamimidoyl-piperidin-4-ylmethyl esterwas also prepared as a TFA salt:

MS m/z: [M+H]⁺calcd for C₂₁H₂₃N₃O₃, 366.17 found 366.2.

Assay 1 Radioligand Binding Assay Membrane Preparation from CellsExpressing hM₁, hM₂, hM₃ and hM₄ Muscarinic Receptor Subtypes

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in medium consisting of HAM's F-12 supplemented with 10% FBSand 250 μg/mL Geneticin. The cells were grown in a 5% CO₂, 37° C.incubator and lifted with 2 mM EDTA in dPBS. Cells were collected by 5minute centrifugation at 650×g, and cell pellets were either storedfrozen at −80° C. or membranes were prepared immediately. For membranepreparation, cell pellets were resuspended in lysis buffer andhomogenized with a Polytron PT-2100 tissue disrupter (Kinematica AG; 20seconds×2 bursts). Crude membranes were centrifuged at 40,000×g for 15minutes at 4° C. The membrane pellet was then resuspended withresuspension buffer and homogenized again with the Polytron tissuedisrupter. The protein concentration of the membrane suspension wasdetermined by the method described in Lowry, O. et al., Journal ofBiochemistry 193:265 (1951). All membranes were stored frozen inaliquots at −80° C. or used immediately. Aliquots of prepared hM₅receptor membranes were purchased directly from Perkin Elmer and storedat −80° C. until use.

Radioligand Binding Assay on Muscarinic Receptor Subtypes hM₂, hM₂, hM₃,hM₄ and hM₅

Radioligand binding assays were performed in 96-well microtiter platesin a total assay volume of 1000 μL. CHO cell membranes stably expressingeither the hM₁, hM₂, hM₃, hM₄ or hM₅ muscarinic subtype were diluted inassay buffer to the following specific target protein concentrations(μg/well): 10 μg for hM₁, 10-15 μg for hM₂, 10-20 μg for hM₃, 10-20 μgfor hM₄, and 10-12 μg for hM₅. The membranes were briefly homogenizedusing a Polytron tissue disruptor (10 seconds) prior to assay plateaddition. Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 40 μM in dilution buffer and then serially diluted 5×with dilution buffer to final concentrations ranging from 400 fM to 4μM. The addition order and volumes to the assay plates were as follows:825 μL it assay buffer with 0.1% BSA, 25 μL radioligand, 100 μL dilutedtest compound, and 50 μL membranes. Assay plates were incubated for 6hours at 37° C. Binding reactions were terminated by rapid filtrationover GF/B glass fiber filter plates (Perkin Elmer Inc., Wellesley,Mass.) pre-treated in 0.3% polyethyleneimine (PEI). Filter plates wererinsed three times with wash buffer (10 mM HEPES) to remove unboundradioactivity. Plates were then air dried, and 50 μL Microscint-20liquid scintillation fluid (PerkinElmer Inc., Wellesley, Mass.) wasadded to each well. The plates were then counted in a PerkinElmerTopcount liquid scintillation counter (PerkinElmer Inc., Wellesley,Mass.). Binding data were analyzed by nonlinear regression analysis withthe GraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the one-site competition model. K_(i) values for testcompounds were calculated from observed IC₅₀ values and the K_(D) valueof the radioligand using the Cheng-Prusoff equation (Cheng Y; Prusoff W.H. Biochemical Pharmacology 22(23):3099-108 (1973)). K_(i) values wereconverted to pK_(i) values to determine the geometric mean and 95%confidence intervals. These summary statistics were then converted backto K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. Exemplary compoundsof the invention that were tested in this assay, were found to have aK_(i) value of less than about 100 nM for the M₃ muscarinic receptorsubtype in this assay. More typically, these compounds were found tohave K_(i) values of less than about 50 nM, with some compounds havingK_(i) values of less than about 10 nM or less than about 1.0 nM.

Assay 2 Muscarinic Receptor Functional Potency Assays Blockade ofAgonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of a test compound is determinedby measuring the ability of the test compound to blockoxotremorine-inhibition of forskolin-mediated cAMP accumulation inCHO-K1 cells expressing the hM₂ receptor.

cAMP assays are performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with ¹²⁵I-cAMP (NENSMP0044B, PerkinElmer Life Sciences Inc., Boston, Mass.), according tothe manufacturer's instructions.

Cells are rinsed once with dPBS and lifted with Trypsin-EDTA solution(0.05% trypsin/0.53 mM EDTA) as described in Assay 1. The detached cellsare washed twice by centrifugation at 650×g for five minutes in 50 mLsdPBS. The cell pellet is then re-suspended in 10 mL dPBS, and the cellsare counted with a Coulter Z1 Dual Particle Counter (Beckman Coulter,Fullerton, Calif.). The cells are centrifuged again at 650×g for fiveminutes and re-suspended in stimulation buffer to an assay concentrationof 1.6×10⁶-2.8×10⁶ cells/mL.

The test compound is initially dissolved to a concentration of 400 μM indilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)), and thenserially diluted with dilution buffer to final molar concentrationsranging from 100 μM to 0.1 nM. Oxotremorine is diluted in a similarmanner.

To measure oxotremorine inhibition of AC activity, 25 μL forskolin (25μM final concentration diluted in dPBS), 25 μL diluted oxotremorine, and50 μL cells are added to agonist assay wells. To measure the ability ofa test compound to block oxotremorine-inhibited AC activity, 25 μLforskolin and oxotremorine (25 μM and 5 μM final concentrations,respectively, diluted in dPBS) 25 μL diluted test compound, and 50 μLcells are added to remaining assay wells.

Reactions are incubated for 10 minutes at 37° C. and stopped by additionof 100 μL ice-cold detection buffer. Plates are sealed, incubatedovernight at room temperature and counted the next morning on aPerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc.,Wellesley, Mass.). The amount of cAMP produced (pmol/well) is calculatedbased on the counts observed for the samples and cAMP standards, asdescribed in the manufacturer's user manual. Data are analyzed bynonlinear regression analysis with the GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.) using the non-linearregression, one-site competition equation. The Cheng-Prusoff equation isused to calculate the K_(i), using the EC₅₀ of the oxotremorineconcentration-response curve and the oxotremorine assay concentration asthe K_(D) and [L], respectively. The K_(i) values are converted topK_(i) values to determine the geometric mean and 95% confidenceintervals. These summary statistics are then converted back to K_(i)values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of the invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of oxotremorine-inhibition offorskolin-mediated cAMP accumulation in CHO-K1 cells expressing the hM₂receptor.

Blockade of Agonist-Mediated [³⁵S]GTPγS Binding

In a second functional assay, the functional potency of test compoundscan be determined by measuring the ability of the compounds to blockoxotremorine-stimulated [³⁵S]GTPγS binding in CHO-K1 cells expressingthe hM₂ receptor.

At the time of use, frozen membranes are thawed and then diluted inassay buffer with a final target tissue concentration of 5-10 μg proteinper well. The membranes are briefly homogenized using a Polytron PT-2100tissue disrupter and then added to the assay plates.

The EC₉₀ value (effective concentration for 90% maximal response) forstimulation of [³⁵S]GTPγS binding by the agonist oxotremorine isdetermined in each experiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following is added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine (EC₉₀) and GDP (3 μM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates are then incubated at 37° C. for 60 minutes. The assayplates are filtered over 1% BSA-pretreated GF/B filters using aPerkinElmer 96-well harvester. The plates are rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) is added to each well, and each plate issealed and radioactivity counted on a topcounter (PerkinElmer). Data areanalyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation is used to calculate the K_(i), using the IC₅₀ values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of the invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of oxotremorine-stimulated [³⁵S]GTPγSbinding in CHO-K1 cells expressing the hM₂ receptor.

Blockade of Agonist-Mediated Calcium Release via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR (Molecular Devices, Sunnyvale, Calif.) assaycapitalizes on this increase in intracellular calcium by using a calciumsensitive dye (Fluo-4AM, Molecular Probes, Eugene, Oreg.) thatfluoresces when free calcium binds. This fluorescence event is measuredin real time by the FLIPR, which detects the change in fluorescence froma monolayer of cells cloned with human M₁ and M₃, and chimpanzee M₅receptors. Antagonist potency can be determined by the ability ofantagonists to inhibit agonist-mediated increases in intracellularcalcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing thehM₁, hM₃ and cM₅ receptors are seeded into 96-well FLIPR plates thenight before the assay is done. Seeded cells are washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4,2 mM calcium chloride, 2.5 mM probenecid in HBSS without calcium andmagnesium) to remove growth media and leaving 50 μL/well of FLIPRbuffer. The cells are then incubated with 50 μL/well of 4 μM FLUO-4AM (a2× solution was made) for 40 minutes at 37° C., 5% carbon dioxide.Following the dye incubation period, cells are washed two times withFLIPR buffer, leaving a final volume of 50 μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine is first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells are first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which is performed by the FLIPR. An EC₉₀ value for oxotremorine isgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100-F)^1/H)*EC₅₀. An oxotremorine concentration of 3×EC_(F)is prepared in stimulation plates such that an EC₉₀ concentration ofoxotremorine is added to each well in the antagonist inhibition assayplates.

The parameters used for the FLIPR are: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline is determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measures thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence is expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data is analyzed againstthe logarithm of drug concentration by nonlinear regression withGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) using thebuilt-in model for sigmoidal dose-response. Antagonist K_(i) values aredetermined by Prism using the oxotremorine EC₅₀ value as the K_(D) andthe oxotremorine EC₉₀ for the ligand concentration according to theCheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. The exemplifiedcompounds of the invention are expected to have a K_(i) value of lessthan about 100 nM for blockade of agonist-mediated calcium release inCHO cells stably expressing the hM₃ receptor.

Assay 3 Rat Einthoven Assay

This in vivo assay is used to assess the bronchoprotective effects oftest compounds exhibiting muscarinic receptor antagonist activity.

All test compounds are diluted in sterile water and are dosed via theinhalation route (IH). The rats (Sprague-Dawley, male, 250-350 g) areexposed to the aerosol generated from an LC Star Nebulizer Set anddriven by a mixture of gases (5% CO₂/95% atmospheric air). Each testcompound solution is nebulized over a 10 minute time period in a pieshaped dosing chamber capable of holding six rats. At predetermined timepoints after inhalation of compound, the Einthoven assay is performed.

Thirty minutes prior to the start of pulmonary evaluation, the animalsare anesthetized with inactin (thiobutabarbital, 120 mg/kg IP). Thejugular vein is catheterized with saline filled polyethylene catheters(PE-50) and used to infuse MCh. The trachea is then dissected andcannulated with a 14 G needle and used for rat ventilation duringpulmonary evaluation. Once surgery is complete, rats are ventilatedusing a piston respirator set at a stroke volume of 1 ml/100 g bodyweight but not exceeding 2.5 ml volume, and at a rate of 90 strokes perminute.

The changes in pressure that occur with each breath are measured.Baseline values are collected for at least 2.5 minutes then rats arechallenged non-cumulatively with 2-fold incremental increases of thebronchoconstrictor MCh (5, 10, 20, 40 and 80 μg/ml). The MCh is infusedfor 2.5 minutes from a syringe pump at a rate of 2 mL/kg/min. Theanimals are euthanized upon completion of the studies.

Changes in ventilation pressure (cm H₂0) in treated animals areexpressed as % inhibition of MCh response relative to control animals.In this assay, a higher % inhibition value indicates that the testcompound has a bronchoprotective effect. Exemplary compounds of theinvention that are tested in this assay at a dose of 100 μg/ml areexpected to exhibit greater than 35% inhibition, some are expected toexhibit greater than 70% inhibition, and some are expected to exhibitgreater than 90% inhibition.

1.5 hr ID₅₀ Determination

Standard muscarinic antagonists were evaluated in the rat Einthovenassay 1.5 hrs post-dose. The order of potency (ID₅₀s) for the fivestandards tested was determined to be: ipratropium (4.4μg/ml)>tiotropium (6 μg/ml)>des-methyl-tiotropium (12μg/ml)>glycopyrrolate (15 μg/ml)>LAS-34237 (24 μg/ml). The potency ofthe test compound is similarly determined at 1.5 hrs post-dose.

6 and 24 hr ID₅₀ Determination

Standards tiotropium and ipratropium were also evaluated 24 hr and/or 6hr post-dose in the rat Einthoven assay. Ipratropium (10 and 30 μg/ml)was about 3-fold less potent 6-hr post-dose compared to its 1.5 hrpotency. The observed loss of activity at this time point (6 hr) isconsistent with its relatively short duration of action in the clinic.Tiotropium showed a slow onset of effect with peak bronchoprotectionbeing achieved 6-hr post-dose. Its 6 hr and 24 hr potency values werenot significantly different from each other and were about 2-fold morepotent compared to its 1.5 hr potency. The onset of action of the testcompound, as well as the 6 and 24 hr potency values, is similarlydetermined.

Assay 4 Rat Antisialagogue Assay

Rats (Sprague-Dawley, male, 250-350 g) are dosed, anesthetized andcannulated as described for Assay 3. At predetermined time points andafter surgery, animals are placed on their dorsal side at a 20° inclinewith their head in a downward slope. A pre-weighed gauze pad is insertedin the animal's mouth and the muscarinic agonist pilocarpine (PILO) (3mg/kg, iv.) is administered. Saliva produced during 10 minutes post-PILOis measured gravimetrically by determining the weight of the gauze padbefore and after PILO. Antisialagogue effects are expressed as %inhibition of salivation relative to control animals.

1, 6 and 24 hr ID₅₀ Determination

The rat antisialagogue assay was developed to assess systemic exposureand calculate the lung selectivity index (LSI) of test compounds. Thestandard, tiotropium, was evaluated in this model at 1, 6, and 24 hrpost-dose. Tiotropium was found to be most potent at inhibitingpilocarpine-induced salivation 6 hrs post dose. This finding isconsistent with the peak effects observed in the Einthoven assay.

This model is a modified version of the procedure described in Rechter,“Estimation of anticholinergic drug effects in mice by antagonismagainst pilocarpine-induced salivation” Ata Pharmacol Toxicol 24:243-254(1996). The mean weight of saliva in vehicle-treated animals, at eachpre-treatment time, is calculated and used to compute % inhibition ofsalivation, at the corresponding pre-treatment time, at each dose.

Exemplary compounds of the invention that are tested in this assay areexpected to exhibit ID₅₀ values less than 100 μg/ml (measured at 24hours), with some compounds expected to exhibit an ID₅₀ value less than30 μg/ml, some less than 20 μg/ml, and some less than 15 μg/ml.

The ratio of the anti-sialagogue ID₅₀ to bronchoprotective ID₅₀ is usedto compute the apparent lung selectivity index of the test compound.Generally, compounds having an apparent lung selectivity index greaterthan about 5 are preferred.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

What is claimed is:
 1. A process for preparing a compound selected from4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-furan-2-ylmethylpiperazine-1-carboxamidineand4-((R)-2-cyclopentyl-2-hydroxy-2-phenylacetyl)-N-thiophen-2-ylmethyl-piperazine-1-carboxamidine,or a pharmaceutically acceptable salt thereof, comprising: (a) couplingcompound (1) and compound (2) under amide bond-forming conditions anddeprotecting the product to form compound (3):

where a is 0; R¹ is cyclopentyl; R² is phenyl; R³ is —OH; and P is anamino-protecting group; (b) reacting compound (3) with compound (6) toform compound (7):

where X is a bond, Y is —CH₂—, Y′ is —N—, Y″ is —CH₂—, and R⁶ is H; and;(c) reacting compound (7) and compound (8) to provide a compound offormula I:

where R⁷ is H, Z is —CH₂-Q, and Q is furanyl or thiophenyl.